Targeting of pharmaceutical agents to pathologic areas using bifunctional fusion polymers

ABSTRACT

Provided herein are new compositions and methods to target pharmaceutical agents to pathological areas by utilizing bifunctional fusion polymers or nanoparticles. These fusion polymers and nanoparticles contain two or more domains: (i) sequences that bind to exposed collagenous (XC-) proteins present in pathological areas, including cancerous lesions and (ii) domains that bind to pharmaceutical agents. The drug-binding functionality of these fusion polymers and nanoparticles is based on high-affinity, non-covalent interactions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 62/056,859, filed on Sep. 29, 2014; 62/103,489, filed on Jan. 14,2015; and 62/214,752, filed on Sep. 4, 2015. The entire contents of theforegoing are incorporated herein by reference.

TECHNICAL FIELD

This invention relates to new compositions and methods capable ofselective and efficient targeting of pharmaceutical agents topathological areas such as cancerous lesions.

BACKGROUND

Cancer is the second most common cause of death in the US, claiming580,000 Americans per year, more than 1,500 people each day. The numberof new cancer patients diagnosed in 2012 was over 1.6 million in theU.S. alone, not including patients with noninvasive cancers and/or skincancers. The National Institutes of Health (NIH) estimated the overallannual costs of cancer care at more than $227 billion (in 2007):including $89 billion for direct medical costs. Sales of cancer drugs ingeneral doubled between 2005 and 2010, with conservative growthestimates of 8 to 10% per year, reaching $93 billion by 2016. Much ofthe overall healthcare costs of treating cancer are derived frommanagement of the deleterious side effects of radiation and conventionalchemotherapy. Nonetheless, the chemotherapy market is currently thefastest growing segment of the pharmaceutical industry, with recentestimates topping $50 billion (in 2012) and rising. Likewise, the globalmarket for therapeutic antibodies (targeted biologics) is estimated torise from $40 billion to $58 billion by the year 2016. However, thesecurrent cancer therapies, including surgery, systemic chemotherapy,radiation therapy, risk factor modification, are often clinicallyinsufficient and/or unacceptably toxic. The systemic toxicities of manyFDA-approved chemotherapeutic agents are a result of the non-specificdistribution of these cytocidal agents in the body, which kills bothcancer cells and normal cells and negatively impacts the treatmentregimen and patient outcome.

SUMMARY

The present disclosure is based, at least in part, on the development ofnew bifunctional fusion polymers that include at least two functionaldomains: (i) sequences that bind to the Exposed Collagenous (XC-)proteins present in pathological areas such as cancerous lesions, and(ii) sequences that bind directly or indirectly to a particular class ofchemotherapeutic or biologic agents, for example, paclitaxel, monoclonalantibodies, growth factors, or small interfering RNA (siRNA). Thepresent disclosure is also based, at least in part, on the developmentof new bifunctional fusion nanoparticles that include at least twoportions: (i) sequences that bind to the Exposed Collagenous (XC-)proteins present in pathological areas, and (ii) a nanoparticle thatnon-covalently sequesters chemotherapeutic and/or biologic agents, forexample agents with hydrophobic or hydrophilic characteristics ornucleic acids. The bifunctional properties of these engineered fusionpolymers and nanoparticles may enable selective and efficient targetingof the widely used chemotherapeutic and biologic agents to abnormal,diseased, or degenerative tissues such as tumors, allowing lower dosesof these agents to become more effective at killing cancer cells andassociated blood supply. Targeting is achieved by combining atumor-targeting functional domain with a high-affinity, non-covalentdrug-binding domain of the fusion polymer or with a nanoparticle of thefusion nanoparticle, generating drug complexes with improvedbiodistribution. Targeted delivery of drugs using the fusion peptide ornanoparticle disclosed herein can reduce systemic toxicity and sideeffects by sequestering the drugs in the tumor microenvironment andsparing normal cells and tissues from the toxicity of the drugs.Moreover, by targeting a common histopathologic property of primarytumors and metastatic lesions, the drug delivery systems describedherein can (i) bind and carry one or more therapeutic drugs, in someinstances upon simple mixing, and (ii) seek out and accumulate in thediseased/cancerous tissues following intravenous infusion. Thus, thefusion peptides and nanoparticles described herein may make conventionalchemotherapeutic and biologic agents more efficient with great efficacywhile lessening unwanted side effects, improving the overall TherapeuticIndex and patient survival. These fusion polymers and nanoparticles canalso include linker segments and/or flanking sequences to improve thefunctionality, pharmacokinetics, stability, and/or pharmacodynamics ofthe targeted drug delivery.

Provided herein are fusion polymers that include (i) an aptamer sequencethat binds to exposed collagenous (XC) proteins present in pathologicalareas, including cancerous lesions, and (ii) an aptamer sequence thatbinds to a drug or biologic agent with therapeutic properties. In someembodiments, the collagen-binding sequence is derived from acollagen-binding domain found in von Willebrand factor, or aconservative variation thereof, which retains collagen-binding activity.For example, the collagen-binding sequence can include the minimaldecapeptide Trp-Arg-Glu-Pro-Ser-Phe-Met-Ala-Leu-Ser (SEQ ID NO: 1) orTrp-Arg-Glu-Pro-Gly-Arg-Met-Glu-Leu-Asn (SEQ ID NO: 2). The drug can bea cytotoxic or cytostatic agent used for chemotherapy (e.g., achemotherapeutic agent). In some embodiments, the drug is a monoclonalantibody or a growth factor. In some embodiments, the drug is an RNA oran siRNA. In some embodiments, a pharmaceutical composition comprises,consists essentially of, or consists of, the fusion polymer and apharmaceutically acceptable carrier. Also included are methods oftreating cancer in a subject that include, e.g., administering to thesubject a pharmaceutically effective amount of a pharmaceuticalcomposition described herein. The cancer can be a primary or metastaticcancer e.g., colorectal cancer, breast cancer, brain cancer, non-smallcell lung cancer, pancreatic cancer, prostate cancer, a sarcoma, acarcinoma, or a melanoma.

In some aspects, the fusion polymers described herein include (i) anaptamer sequence that binds to XC proteins present in solid tumors, and(ii) an aptamer sequence that binds to a drug, e.g., an angiogenesismodulating agent, e.g., for directly or indirectly affecting endothelialcell and/or tumor cell proliferation. The angiogenesis modulating agentcan be selected from: growth factors including vascular endothelialgrowth factor (VEGF), epidermal growth factor (EGF), hepatocyte growthfactor (HGF), platelet derived endothelial cell growth factor (PD-ECGF),platelet derived growth factor (PDGF); insulin-like growth factor (IGF),interleukin-8, growth hormone, angiopoietin, acidic and basic fibroblastgrowth factors (FGFs), transforming growth factor alpha (TGF-alpha.), anenzyme, an enzymatic inhibitor, and/or an antibody specific for thesegrowth factors and their receptors.

In some embodiments, a pharmaceutical composition comprises, consistsessentially of, or consists of, the fusion polymer and apharmaceutically acceptable carrier. Also included are methods oftreating cancer in a subject that include, e.g., administering to thesubject a pharmaceutically effective amount of the pharmaceuticalcomposition described herein. The cancer can be a primary or metastaticcancer as described herein.

In some embodiments, the angiogenesis modulating agent is a monoclonalantibody (mAb) and the resulting onco-aptamer is referred to as amAb-Tropin or mAb/onco-aptamer. In some embodiments, the angiogenesismodulating agent is directed against VEGF or specifically binds VEGF,for example, the angiogenesis modulating agent can be bevacizumab,aflibercept, or rilonacept. In some embodiments, the angiogenesismodulating agent can inhibit or trap VEGF when VEGF passes through acollagen-agarose column that contains collagen-bound onco-aptamer/mAbcomplexes. In some embodiments, the angiogenesis modulating agentinhibits vascular endothelial cell proliferation. In some embodiments,the mAb-Tropins or mAb/onco-aptamers are injected systemically to asubject and bind to exposed collagenous (XC) proteins found abundantlyin tumors undergoing tumor invasion, metastasis, stroma formation andneoangiogenesis. In some embodiments, the mAb-Tropins ormAb/onco-aptamers are injected systemically to inhibit tumor growth byinhibiting tumor endothelial cell proliferation. In some embodiments,the mAb-Tropins or mAb/onco-aptamers are injected systemically for thetreatment of a wide variety of primary and metastatic cancers, includingbut not restricted to colorectal cancer, breast cancer, brain tumors,sarcoma, carcinoma, and melanoma. In some embodiments, a pharmaceuticalcomposition comprises, consists essentially of, or consists of, thefusion polymer and a pharmaceutically acceptable carrier.

In some embodiments, the angiogenesis modulating agent is achemotherapeutic agent used in a metronomic regimen to induceanti-angiogenesis.

In some embodiments the aptamer that binds to a drug, or drug bindingdomain, is a chemotherapeutic agent binding domain and the drug is achemotherapeutic agent. For example, the chemotherapeutic agent can be,but is not restricted to, paclitaxel, docetaxel, or nab-paclitaxel, andthe resulting onco-aptamer is referred to as Taxol-Tropin orAdaptane-Tx. In some embodiments, a pharmaceutical compositioncomprises, consists essentially of, or consists of, the fusion polymer,the chemotherapeutic agent and a pharmaceutically acceptable carrier. Insome embodiments, Taxol-Tropin or Adaptane-Tx is injected systemicallyto a subject to inhibit tumor growth by inhibiting tumor endothelialcell proliferation. In some embodiments, Taxol-Tropin or Adaptane-Tx isinjected systemically to a subject for the treatment of a wide varietyof primary and metastatic cancers, including but not restricted tocolorectal cancer, breast cancer, brain tumors, non-small cell lungcancer, pancreatic cancer and prostate cancer.

In some embodiments, the drug binding domain of the fusion polymer is achemotherapeutic agent binding domain, the drug is a chemotherapeuticagent and the chemotherapeutic agent is a monoclonal antibody (mAb). Insome embodiments, the chemotherapeutic agent comprises, consistsessentially of, or consists of a mAb that is selected from the groupconsisting of an anti-CTLA-4, anti-PD-1, and anti-PD-L1 antibody. Insome embodiments, the mAb is Ipililumab, Nivolumab, Pembrolizumab, orany combination thereof. In some embodiments, a pharmaceuticalcomposition comprises, consists essentially of, or consists of, thefusion polymer, a mAb and a pharmaceutically acceptable carrier. Alsoprovided are methods of treating cancer in a subject, the methodcomprising, consisting essentially of, or consisting of: administeringto a subject in need of such treatment the fusion polymer comprising (a)a collagen-binding domain comprising an amino acid sequence, and (b) amAb-binding domain in an amount sufficient to treat the tumor; and a mAbbound by the mAb-binding domain, wherein the mAb bound to the fusionpolymer activates the immune response within the tumor. The cancer canbe a primary or metastatic cancer as described herein.

In some embodiments, the drug or biologic agent is a siRNAs and theresulting fusion polymer is referred to as RNA-Tropin. In someembodiments, the siRNA-binding domain comprises, consists essentiallyof, or consists of the amino acid sequence of SEQ ID NO: 29 or SEQ IDNO: 30. In some embodiments, the RNA-Tropin fusion polymer comprises,consists essentially of, or consists of the amino acid sequence of SEQID NO: 35, SEQ ID NO: 36, SEQ ID NO:37, SEQ ID NO: 38, or SEQ ID NO: 55.In some embodiments, a pharmaceutical composition comprises, consistsessentially of, or consists of, the fusion polymer, the RNA and apharmaceutically acceptable carrier. In some embodiments, a subject canbe treated with the RNA-Tropin, wherein it is injected systemically forthe treatment of a wide variety of primary and metastatic cancers,including but not restricted to colorectal cancer, breast cancer, braintumors, non-small cell lung cancer, pancreatic cancer, prostate cancer,sarcoma, carcinoma, and melanoma.

In some embodiments, the bifunctional fusion polymer binds to collagenand paclitaxel. The fusion polymer can comprise, consist essentially of,or consist of, (a) a collagen-binding domain comprising an amino acidsequence of SEQ ID NO: 1 or SEQ ID NO: 2; and (b) a paclitaxel-bindingdomain comprising an amino acid sequence selected from the groupconsisting of:Arg-Gly-Val-Gly-Ile-Met-Lys-Ala-Cys-Gly-Arg-Thr-Arg-Val-Thr-Ser (SEQ IDNO: 3);Arg-Gly-Val-Gly-Ile-Met-Lys-Ala-Cys-Gly-Arg-Thr-Arg-His-Thr-Val-Arg (SEQID NO: 4);Arg-Gly-Val-Gly-Ile-Met-Lys-Ala-Cys-Gly-Arg-Thr-Arg-His-Thr-Val-Arg-(D-Met)-Gly(SEQ ID NO: 5); andAla-Phe-Met-Thr-Lys-Thr-Met-Glu-Cys-Gly-Arg-Thr-Arg-His-Thr-Val-Arg-Met-Gly(SEQ ID NO: 6). In some embodiments, the fusion polymer further includesone or more linkers/spacers. For example, the fusion polymer can haveone or more linkers comprising an amino acid sequence such asGly-Gly-Ser-Gly (SEQ ID NO: 7), Arg-Arg-Gly-Val-His-Val-Gly (SEQ ID NO:8), (D-Arg)-(D-Arg)-Gly-Val-His-Val-Gly (SEQ ID NO: 9), orLys-Gly-Arg-Arg-Gly-Val-His-Val-Gly (SEQ ID NO: 10), or variants ofthese sequences. In some embodiments, the fusion polymer is acetylated,amidated, and/or PEGylated at N- or C-terminus. In some embodiments, apharmaceutical composition comprises, consists essentially of, orconsists of, the fusion polymer and a pharmaceutically acceptablecarrier. Also included are methods of treating cancer in a subject thatinclude, e.g., administering to the subject a pharmaceutically effectiveamount of the pharmaceutical composition described herein. The cancercan be a primary or metastatic cancer as described herein.

In some embodiments, the collagen-binding and paclitaxel-binding fusionpolymer comprises, consists essentially of, or consists of, an aminoacid sequence selected from the group consisting of:Arg-Arg-Gly-Val-His-Val-Gly-Trp-Arg-Glu-Pro-Ser-Phe-Met-Ala-Leu-Ser-Met-Pro-His-Gly-Gly-Ser-Gly-Arg-Gly-Val-Gly-Ile-Met-Lys-Ala-Cys-Gly-Arg-Thr-Arg-Val-Thr-Ser-Ala-Gly-Ser-Gly(SEQ ID NO: 11);(D-Arg)-(D-Arg)-Gly-Val-His-Val-Gly-Trp-Arg-Glu-Pro-Gly-Arg-Met-Glu-Leu-Asn-Met-Pro-His-Gly-Gly-Ser-Gly-Arg-Gly-Val-Gly-Ile-Met-Lys-Ala-Cys-Gly-Arg-Thr-Arg-His-Thr-Val-Arg-(D-Met)-Gly(SEQ ID NO: 12);Lys-Gly-Arg-Arg-Gly-Val-His-Val-Gly-Trp-Arg-Glu-Pro-Gly-Arg-Met-Glu-Leu-Asn-Met-Pro-His-Gly-Gly-Ser-Gly-Arg-Gly-Val-Gly-Ile-Met-Arg-Ala-Cys-Gly-Arg-Thr-Arg-His-Thr-Val-Arg-(D-Met)-Gly(SEQ ID NO: 13), and(D-Arg)-(D-Arg)-Gly-Val-His-Val-Gly-Trp-Arg-Glu-Pro-Gly-Arg-Met-Glu-Leu-Asn-Met-Pro-His-Gly-Gly-Ser-Gly-Arg-Gly-Val-Gly-Ile-Met-Lys-Ala-Cys-Gly-Arg-Thr-Arg-His-Thr-Val-Arg-Met-Gly-(D-Pro)-(D-Thr) (SEQ ID NO: 14).

In some embodiments, the bifunctional fusion polymer binds to collagenand a therapeutic immunoglobulin such as a mAb. The fusion polymer cancomprise, consist essentially of, or consist of, (a) a collagen-bindingdomain comprising an amino acid sequence of SEQ ID NO: 1, or SEQ ID NO:2; and (b) an immunoglobulin-binding domain comprising the amino acidsequence of His-Trp-Arg-Gly-Trp-Val (SEQ ID NO: 15). In someembodiments, the fusion polymer further includes one or morelinkers/spacers. For example, the fusion polymer can have one or morelinkers comprising, consisting essentially of, or consisting of an aminoacid sequence such as Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 16),Gly-Gly-Ser-Gly-Gly (SEQ ID NO: 17), SEQ ID NO: 8, SEQ ID NO: 9,Cys-Gly-Arg-Arg-Gly-Val-His-Val-Gly (SEQ ID NO: 57), andCys-Ala-Arg-Arg-Gly-Val-His-Val-Gly (SEQ ID NO: 58), or variants ofthese sequences. In some embodiments, the fusion polymer is acetylated,amidated, and/or PEGylated at N- or C-terminus. In some embodiments, apharmaceutical composition comprises, consists essentially of, orconsists of, the fusion polymer and a pharmaceutically acceptablecarrier. Also included are methods of treating cancer in a subject thatinclude, e.g., administering to the subject a pharmaceutically effectiveamount of the pharmaceutical composition described herein. The cancercan be a primary or metastatic cancer as described herein.

In some embodiments, the collagen-binding and immunoglobulin-bindingfusion polymer comprises, consists essentially of, or consists of, anamino acid sequence selected from the group consisting of:Arg-Arg-Gly-Val-His-Val-Gly-Trp-Arg-Glu-Pro-Ser-Phe-Met-Ala-Leu-Ser-Met-Pro-His-Gly-Gly-Gly-Gly-Gly-His-Trp-Arg-Gly-Trp-Val-Gly-Gly-Gly-Gly-Gly-His-Trp-Arg-Gly-Trp-Val(SEQ ID NO: 18);(D-Arg)-(D-Arg)-Gly-Val-His-Val-Gly-Trp-Arg-Glu-Pro-Gly-Arg-Met-Glu-Leu-Asn-Met-Pro-His-Gly-Gly-Ser-Gly-Gly-His-Trp-Arg-Gly-Trp-Val-Ala-Gly-Gly-Ser-Gly-Gly-His-Trp-Arg-Gly-Trp-Val-(D-Ala)-(D-Ala)(SEQ ID NO: 19);Cys-Gly-Arg-Arg-Gly-Val-His-Val-Gly-Trp-Arg-Glu-Pro-Gly-Arg-Met-Glu-Leu-Asn-Met-Pro-His-Gly-Gly-Ser-Gly-Gly-His-Trp-Arg-Gly-Trp-Val-Ala-Gly-Gly-Ser-Gly-Gly-His-Trp-Arg-Gly-Trp-Val-(D-Ala)-(D-Ala)(SEQ ID NO: 20); andCys-Ala-Arg-Arg-Gly-Val-His-Val-Gly-Trp-Arg-Glu-Pro-Gly-Arg-Met-Glu-Leu-Asn-Met-Pro-His-Gly-Gly-Ser-Gly-Gly-His-Trp-Arg-Gly-Trp-Val-Ala-Gly-Gly-Ser-Gly-Gly-His-Trp-Arg-Gly-Trp-Val-Ala-(D-Pro)-(D-Thr) (SEQ ID NO: 21).

In some embodiments, the bifunctional fusion polymer binds to collagenand a therapeutic ribonucleic acid such as a siRNA. This fusion polymercomprises, consists essentially of, or consists of, (a) acollagen-binding domain comprising an amino acid sequence of SEQ ID NO:1 or SEQ ID NO: 2; and (b) an RNA-binding domain comprising an aminoacid sequence of Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg (SEQ ID NO: 29), or(D-Arg)-(D-Arg)-(D-Arg)-(D-Arg)-(D-Arg)-(D-Arg)-(D-Arg)-(D-Arg)-(D-Arg)(SEQ ID NO: 30). In some embodiments, the fusion polymer furtherincludes one or more linkers/spacers. For example, the fusion polymercan have one or more linkers comprising an amino acid sequence such asGly-Gly-Ser-Gly-Gly-Ser-Gly-Gly (SEQ ID NO: 31),Gly-Gly-Arg-Arg-Gly-Val-His-Val-Gly (SEQ ID NO: 32), SEQ ID NO: 8, SEQID NO: 58, Cys-Gly-Ser-Gly-Gly (SEQ ID NO: 33), and Gly-Gly (SEQ ID NO:34), or variants of these sequences. In some embodiments, the fusionpolymer is amidated, acetylated and/or PEGylated at N- or C-terminus. Insome embodiments, a pharmaceutical composition comprises, consistsessentially of, or consists of, the fusion polymer and apharmaceutically acceptable carrier. Also included are methods oftreating cancer in a subject that include, e.g., administering to thesubject a pharmaceutically effective amount of the pharmaceuticalcomposition described herein. The cancer can be a primary or metastaticcancer as described herein.

In some embodiments, the collagen-binding and RNA-binding fusion polymercomprises, consists essentially of, or consists of, an amino acidsequence selected from the group consisting of:Arg-Arg-Gly-Val-His-Val-Gly-Trp-Arg-Glu-Pro-Ser-Phe-Met-Ala-Leu-Ser-Met-Pro-His-Gly-Gly-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg(SEQ ID NO: 35);Cys-Ala-Arg-Arg-Gly-Val-His-Val-Gly-Trp-Arg-Glu-Pro-Gly-Arg-Met-Glu-Leu-Asn-Met-Pro-His-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-(D-Arg)-(D-Arg)-(D-Arg)-(D-Arg)-(D-Arg)-(D-Arg)-(D-Arg)-(D-Arg)-(D-Arg)(SEQ ID NO: 36);(D-Arg)-(D-Arg)-(D-Arg)-(D-Arg)-(D-Arg)-(D-Arg)-(D-Arg)-(D-Arg)-(D-Arg)-Gly-Gly-Arg-Arg-Gly-Val-His-Val-Gly-Trp-Arg-Glu-Pro-Gly-Arg-Met-Glu-Leu-Asn-Met-Pro-His-Gly-(D-G1n)-(D-Pro)-(D-Thr)(SEQ ID NO: 37);Cys-Gly-Ser-Gly-Gly-(D-Arg)-(D-Arg)-(D-Arg)-(D-Arg)-(D-Arg)-(D-Arg)-(D-Arg)-(D-Arg)-(D-Arg)-Gly-Gly-Arg-Arg-Gly-Val-His-Val-Gly-Trp-Arg-Glu-Pro-Gly-Arg-Met-Glu-Leu-Asn-Met-Pro-His-Gly-(D-G1n)-(D-Pro)-(D-Thr) (SEQ ID NO: 38); andArg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Gly-Val-His-Val-Gly-Trp-Arg-Glu-Pro-Ser-Phe-Met-Ala-Leu-Ser-Met-Pro-His-Gly-Gly(SEQ ID NO: 55).

In some embodiments, the bifunctional fusion polymer binds to collagenand human serum albumin (HSA), wherein the HSA can in turn bind atherapeutic agent. The fusion polymer comprises, consists essentiallyof, or consists of, (a) a collagen-binding domain comprising an aminoacid sequence of SEQ ID NO: 1 or SEQ ID NO: 2; and (b) a human serumalbumin (HSA)-binding domain that comprises a thiol-reactive maleimidegroup, or a peptide comprising the amino acid sequence ofThr-Arg-Ser-Phe-Cys-Thr-Asp-Trp-Pro-Ala-His-Lys-Ser-Cys-Lys-Pro-Leu (SEQID NO: 39), orArg-Gln-Met-Glu-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp-Gly-Asp (SEQID NO: 40). In some embodiments, the fusion polymer further includes oneor more linkers/spacers as described herein, or variants of thesesequences. In some embodiments, the fusion polymer is acetylated,amidated, and/or PEGylated at N- or C-terminus. In some embodiments, thefusion polymer comprises, consists essentially of, or consists of, (a) acollagen-binding tumor-targeting domain comprising an amino acidsequence of SEQ ID NO: 1 or SEQ ID NO: 2; and (b) a HSA-binding domain,wherein the HSA-binding domain comprises a thiol-reactive maleimidegroup covalently linked to the collagen-binding domain or a targetingpeptide comprising the albumin-binding amino acid sequence of SEQ ID NO:39 or SEQ ID NO: 40. In some embodiments, the therapeutic agent thatbinds to the HSA is a platinum drug. In some embodiments, apharmaceutical composition comprises, consists essentially of, orconsists of, the fusion polymer and a pharmaceutically acceptablecarrier. Also included are methods of treating cancer in a subject thatinclude, e.g., administering to the subject a pharmaceutically effectiveamount of the pharmaceutical composition described herein. The cancercan be a primary or metastatic cancer as described herein.

In some embodiments, the collagen-binding and HSA-binding fusion polymercomprises, consists essentially of, or consists of, an amino acidsequence selected from the group consisting of:(D-Arg)-(D-Arg)-Gly-Val-His-Val-Gly-Trp-Arg-Glu-Pro-Gly-Arg-Met-Glu-Leu-Asn-Met-Pro-His-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Thr-Arg-Ser-Phe-Cys-Thr-Asp-Trp-Pro-Ala-His-Lys-Ser-Cys-Lys-Pro-Leu-(D-Arg)-(D-Ala)(SEQ ID NO: 41);(D-Arg)-(D-Arg)-Gly-Val-His-Val-Gly-Trp-Arg-Glu-Pro-Gly-Arg-Met-Glu-Leu-Asn-Met-Pro-His-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Arg-Gln-Met-Glu-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp-Gly-Asp-(D-Glu)-(D-Asp) (SEQ ID NO: 42);Maleimide-PEG2-Ser-Gly-Gly-Ser-Gly-Ala-Arg-Arg-Gly-Val-His-Val-Gly-Trp-Arg-Glu-Pro-Gly-Arg-Met-Glu-Leu-Asn-Met-Pro-His-Gly-(D-G1n)-(D-Pro)-(D-Thr)(SEQ ID NO: 43), and(D-Ala)-(D-Arg)-(D-Arg)-Gly-Val-His-Val-Gly-Trp-Arg-Glu-Pro-Gly-Arg-Met-Glu-Leu-Asn-Met-Pro-His-Gly-Gly-Ser-Gly-Gly-Ser-Lys-PEG2-Maleimide(SEQ ID NO: 44).

In some embodiments, the collagen-binding and drug-binding fusionpolymer further comprises a HSA-binding domain, e.g., as a thirdportion. The HSA-binding domain comprises, consists essentially of, orconsists of, an amino acid sequence selected from the group consistingof SEQ ID NO: 39 or SEQ ID NO: 40. In some embodiments, the fusionpolymer further includes one or more linker/spacers. For example, thefusion polymer can have one or more linkers wherein the one or morelinkers connect the HSA-binding, XC-binding, and the drug-bindingdomains or are connected to the terminus of the polymer. In someembodiments, the fusion polymer is acetylated, amidated and/or PEGylatedat N- or C-terminus. In some embodiments, the drug-binding domain is achemotherapeutic agent binding domain and the drug is a chemotherapeuticagent. In some embodiments, the chemotherapeutic agent is paclitaxel,docetaxel, or nab-paclitaxel. In some embodiments, a pharmaceuticalcomposition comprises, consists essentially of, or consists of, thefusion polymer and a pharmaceutically acceptable carrier. Also includedare methods of treating cancer in a subject. The methods include, e.g.,administering to the subject a pharmaceutically effective amount of thepharmaceutical composition described herein. The cancer can be a primaryor metastatic cancer as described herein.

In some embodiments, the fusion polymer further comprises a PEG moietyor a thio-reactive maleimide group, wherein the PEG moiety or thethio-reactive group is linked to the N- or C-terminus of the polymer.For example, the fusion polymer can comprise, consist essentially of, orconsist of, an amino acid sequence ofminiPEG-Ser-Gly-Gly-Ser-Gly-Ala-Arg-Arg-Gly-Val-His-Val-Gly-Trp-Arg-Glu-Pro-Gly-Arg-Met-Glu-Leu-Asn-Met-Pro-His-Gly-Gly-Ser-Gly-Arg-Gly-Val-Gly-Ile-Met-Lys-Ala-Cys-Gly-Arg-Thr-Arg-His-Thr-Val-Arg-(D-Met)-Gly-(D-Ser) (SEQ ID NO: 27), wherein (D-Aaa)designates D-amino acids. The fusion polymer can further comprise one ormore linkers, where the one or more linkers join the differentfunctional domains (e.g., the HSA-binding domain, the XC-binding domain,or the drug-binding domain) or are connected to the terminus of thepolymer. The one or more linkers comprises, consists essentially of, orconsists of, an amino acid sequence selected from the group consistingof Gly-Gly-Gly-Gly (SEQ ID NO: 22), SEQ ID NO: 7, Ser-Gly-Gly-Ser-Gly(SEQ ID NO: 23), SEQ ID NO: 17, Gly-Ser-Gly-Ser-Gly-Ser (SEQ ID NO: 24),Gly-Gly-Ser-Gly-Gly-Ser-Lys (SEQ ID NO: 25), SEQ ID NO: 31, andGly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly (SEQ ID NO: 26), wherein the one ormore linkers connect the XC-binding and drug-binding domains or areconnected to the terminus of the polymer. In some embodiments, apharmaceutical composition comprises, consists essentially of, orconsists of, the fusion polymer and a pharmaceutically acceptablecarrier. Also included are methods of treating cancer in a subject thatinclude, e.g., administering to the subject a pharmaceutically effectiveamount of the pharmaceutical composition described herein. The cancercan be a primary or metastatic cancer as described herein.

In some aspects, the fusion polymer comprises a tumor-targeting sequencethat binds to exposed collagen (XC) proteins, and a di-block copolymerthat comprises a distinct hydrophilic block and a distinct hydrophobicblock. The fusion polymer can further comprise one or more linkers. Theone or more linkers can comprise, consist essentially of, or consist of,an amino acid sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO: 7, SEQ ID NO: 23, SEQ ID NO: 17, SEQ ID NO: 24, SEQ IDNO: 25, SEQ ID NO: 31, SEQ ID NO: 26 and SEQ ID NO: 9, wherein a firstlinker is bound to the N-terminus of the tumor-targeting aptamer andfurther comprises a cysteine residue on the N-terminus of the firstlinker, and a second linker connects the hydrophilic terminus of thedi-block to a maleimide-group, wherein the cysteine residue of the firstlinker and the maleimide-group of the second linker are covalentlylinked, thereby connecting the tumor-targeting aptamer and the di-blockcopolymer, making the fusion polymer. In some embodiments, apharmaceutical composition comprises, consists essentially of, orconsists of, the fusion polymer and a pharmaceutically acceptablecarrier. Also included are methods of treating cancer in a subject thatinclude, e.g., administering to the subject a pharmaceutically effectiveamount of the pharmaceutical composition described herein. The cancercan be a primary or metastatic cancer as described herein.

In some embodiments the hydrophilic block comprises, consistsessentially of, or consists of, a hydrophilic polymer. Exemplaryhydrophilic polymers include poly-ethylene glycol (PEG), poly-N-vinylpyrrolidone (PVP), and poly-N-isopropyl acrylamide (pNIPAM or NIPAM), orother variants (see Sutton et al., Pharmaceutical Res., 2007,24:1029-1045 incorporated herein in its entirety).

In some embodiments the hydrophobic block comprises, consistsessentially of, or consists of, a hydrophobic polymer. Exemplaryhydrophobic polymers include alkyl chain, poly-L-lactide (PLLA),poly-D,L-lactide (PDLLA), poly-caprolactone (PCL),poly-D,L-lactic-co-glycolic acid (PLGA), poly-delta-verolactone (PVL),and poly-L-histadine (pHis) (see Shuai et al., 2004, J ControlledRelease, 98:415-426; Sutton et al., Pharmaceutical Res., 2007,24:1029-1045 which are both incorporated herein in their entirety).

In some embodiments the fusion polymer comprises, consists essentiallyof, or consists of, the di-block copolymer that comprisesMaleimide-[PEG],-[PDLLA]m, wherein n is at least 1 and m is at least 1.In some embodiments the tumor targeting aptamer sequence comprises,consists essentially of, or consists of the amino acid sequencedselected from the group consisting ofCys-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Ala-Arg-Arg-Gly-Val-His-Val-Gly-Trp-Arg-Glu-Pro-Gly-Arg-Met-Glu-Leu-Asn-Met-Pro-His-Gly-(D-G1n)-(D-Pro)-(D-Thr)-amide(SEQ ID NO: 56) oracetyl-(D-Ala)-(D-Arg)-(D-Arg)-Gly-Val-His-Val-Gly-Trp-Arg-Glu-Pro-Gly-Arg-Met-Glu-Leu-Asn-Met-Pro-His-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Cys(SEQ ID NO: 28), wherein (D-Aaa) designate D-amino acids.

Also provided herein are nanoparticles or bifunctional fusionnanoparticles, e.g., micelles and/or liposomes that include the fusionpolymers described herein. In some aspects, a nanoparticle of thepresent invention comprises, consists essentially of, or consists of, amicelle having a hydrophobic interior and a hydrophilic surface, andthat includes at least one fusion polymer that comprises acollagen-binding domain and a di-block copolymer and wherein the tumortargeting domain of the at least one fusion polymer extends outwardlyfrom the hydrophilic surface of the micelle. This arrangement allows thetumor targeting domain extending from the micelle surface to bind to XCproteins while associated with the micelle. In some embodiments, themicelle comprises, consists essentially of, or consists of, a pluralityof the fusion polymers and wherein the fusion polymers comprise at leastor about 1/10^(th) (at least or about 2/10^(th), 3/10^(th), 4/10^(th),5/10^(th), 6/10^(th), 7/10^(th), 8/10^(th) or at least or about9/10^(th), e.g., 10/10^(th)) of the total number of polymers thatcomprise the micelle. In some embodiments, the tumor targeting domaincomprises, consists essentially of, or consists of, an amino acidsequence of SEQ ID NO: 1 or SEQ ID NO: 2 and in some embodiments, thetumor targeting domain binds to exposed collagenous proteins that arepresent in solid tumors. In some embodiments, a pharmaceuticalcomposition comprises, consists essentially of, or consists of, thefusion nanoparticle and a pharmaceutically acceptable carrier. Alsoincluded are methods of treating cancer in a subject that include, e.g.,administering to the subject a pharmaceutically effective amount of thepharmaceutical composition described herein. The cancer can be a primaryor metastatic cancer as described herein.

In some aspects, a drug-delivery or bifunctional fusion nanoparticlecomprises, consists essentially of, or consists of, a micelle comprisingat least or about one fusion polymer and a drug sequestered inside thehydrophobic interior of the micelle. In some embodiments, apharmaceutical composition comprises, consists essentially of, orconsists of, the fusion nanoparticle and a pharmaceutically acceptablecarrier. Also included are methods of treating cancer in a subject thatinclude, e.g., administering to the subject a pharmaceutically effectiveamount of the pharmaceutical composition described herein. The cancercan be a primary or metastatic cancer as described herein.

In some embodiments, the drug sequestered inside the micelle comprises,consists essentially of, or consists of, a drug such as a hydrophobicdrug, a hydrophilic drug, a nucleic acid, a Taxane, doxorubicin,epirubicin, a platinum drug, R547, a cyclin-dependent kinase inhibitor,TGX-221, a PI-3 kinase inhibitor, captothecin, gemcitabine,5-fluouracil, rifampicin, tamoxifen, ellipticin, ethotrexate,daunomycin, estrogen, curcumin, and an siRNA, or any mixture thereof. Insome embodiments the drug is a hydrophobic drug that is sequestered inthe hydrophobic interior of the micelle.

Also provided are methods of making a fusion polymer that comprises atumor targeting aptamer sequence that binds to exposed collagenous (XC)proteins, and a di-block copolymer comprising a hydrophilic block and ahydrophobic block, comprising: (a) providing the tumor targeting aptamersequence and a first linker that is bound to the N-terminus of thetumor-targeting aptamer and further comprises a cysteine residue on theN-terminus of the first linker; (b) providing the di-block copolymer anda second linker that is disposed between and linked to both the terminushydrophilic block of the di-block and a maleimide-group; and (c)combining (a) and (b) under conditions such that the maleimide-group of(b) reacts with the thiol group of the cysteine residue of (a) to form acovalent bond between (a) and (b), thereby creating the fusion polymer.

Also provided are methods of making a micelle that comprises, consistsessentially of, or consists of a hydrophobic interior and a hydrophilicsurface and at least one fusion polymer comprising (i) a tumor targetingdomain that binds to exposed collagenous (XC) proteins, and (ii) adi-block copolymer comprising a hydrophilic block and a hydrophobicblock, wherein the tumor targeting domain of the at least one fusionpolymer extends outwardly from the hydrophilic surface of the micelle,such that the tumor targeting domain can bind to XC proteins whileassociated with the micelle. The method of making a micelle cancomprise: (a) providing a first and a second plurality of di-blockcopolymers, each comprising a hydrophobic block and a hydrophilic block,wherein the di-block copolymers of the second plurality each comprise alinker bound to the terminus of the hydrophilic block, and wherein amaleimide group is bound to the linker terminus that is unbound to thehydrophilic block; (b) mixing the first and second plurality of di-blockcopolymers to thereby form a micelle; (c) providing a tumor-targetingdomain that binds to exposed collagenous proteins and that comprises acysteine residue on a terminus of the domain; and (d) combining themicelle of (b) with the tumor-targeting domain of (c) under conditionssuch that an activated maleimide of the di-block copolymers of thesecond plurality in the micelle reacts with the cysteine residue of thetumor-targeting domain, thereby connecting the tumor-targeting domainand the micelle.

In some embodiments, the method of making a micelle comprises: (a)providing a first and a second plurality of di-block copolymers, eachcomprising a hydrophobic block and a hydrophilic block, wherein thedi-block copolymers of the second plurality each comprise a linker boundto the terminus of the hydrophilic block, and wherein a maleimide groupis bound to the linker terminus that is unbound to the hydrophilicblock; (b) providing a tumor-targeting domain that binds to exposedcollagenous proteins and that comprises a cysteine residue on a terminusof the domain; (c) combining the second plurality of di-block copolymersof (a) with the tumor-targeting domain of (b) under conditions such thatthe activated maleimide of the di-block copolymers reacts with thecysteine residue of the tumor-targeting domain, thereby connecting thetumor-targeting domain and the di-block copolymer and creating a fusionpolymer; and (d) mixing the fusion polymer of (c) with the firstplurality of di-block copolymers to thereby form a micelle.

In some embodiments, the method of making a micelle further comprises,during mixing, adding a therapeutic agent, e.g., an agent describedherein, such as an anticancer agent, under conditions sufficient to forma micelle that encapsulates the therapeutic agent, to thereby make adrug-delivery nanoparticle. In some embodiments, the therapeutic agentis a hydrophobic drug that is sequestered in the hydrophobic interior ofthe micelle.

Also provided are methods of making a drug-delivery nanoparticle thatcomprises, consists essentially of, or consists of (1) a micellecomprising a hydrophobic interior and a hydrophilic surface and at leastone fusion polymer comprising (i) a tumor targeting domain that binds toexposed collagenous (XC) proteins, and (ii) a di-block copolymercomprising a hydrophilic block and a hydrophobic block, wherein thetumor targeting domain of the at least one fusion polymer extendsoutwardly from the hydrophilic surface of the micelle, such that thetumor targeting domain can bind to XC proteins while associated with themicelle; and (2) a drug sequestered in the hydrophobic interior of themicelle, comprising (a) providing the drug; (b) providing the micelle;and (c) mixing the drug and the micelle to allow association of themicelle with the drug, thereby forming a drug-delivery nanoparticle.

Also provided is a fusion polymer that comprises, consists essentiallyof, or consists of, a tumor targeting aptamer sequence that binds toexposed collagenous (XC) protein, and a multi-block polymer comprisingsequentially a first hydrophilic block, a first hydrophobic block, asecond hydrophobic block, and a second hydrophilic block. The fusionpolymer can further comprise one or more linkers as described herein,wherein the one or more linkers link the tumor targeting aptamer and themulti-block polymer or are attached to the terminus of the polymer. Insome embodiments, the linkers comprise, consist essentially of, orconsist of an amino acid sequence selected from the group consisting ofSEQ ID NO: 22, SEQ ID NO: 7, SEQ ID NO: 23, SEQ ID NO: 17, SEQ ID NO:24, SEQ ID NO: 25, SEQ ID NO: 31, SEQ ID NO: 26 and SEQ ID NO: 9. Insome embodiments, the multi-block polymer comprisesMaleimide-[PEG]_(n)-[PDLLA]_(m)-[PDLLA]_(m)-[PEG]_(n), wherein n is atleast 1 and m is at least 1. In some embodiments, a pharmaceuticalcomposition comprises, consists essentially of, or consists of, thefusion polymer and a pharmaceutically acceptable carrier. Also includedare methods of treating cancer in a subject that include, e.g.,administering to the subject a pharmaceutically effective amount of thepharmaceutical composition described herein. The cancer can be a primaryor metastatic cancer as described herein.

In some embodiments the hydrophilic block comprises, consistsessentially of, or consists of, a hydrophilic polymer. Exemplaryhydrophilic polymers include poly-ethylene glycol (PEG), poly-N-vinylpyrrolidone (PVP), and poly-N-isopropyl acrylamide (pNIPAM or NIPAM), orother variants (see Sutton et al., Pharmaceutical Res., 2007,24:1029-1045 incorporated herein in its entirety).

In some embodiments the hydrophobic block comprises, consistsessentially of, or consists of, a hydrophobic polymer. Exemplaryhydrophobic polymers include alkyl chain, poly-L-lactide (PLLA),poly-D,L-lactide (PDLLA), poly-caprolactone (PCL),poly-D,L-lactic-co-glycolic acid (PLGA), poly-delta-verolactone (PVL),and poly-L-histadine (pHis) (see Shuai et al., 2004, J ControlledRelease, 98:415-426; Sutton et al., Pharmaceutical Res., 2007,24:1029-1045 which are both incorporated herein in their entirety).

In some embodiments a bifunctional fusion nanoparticle comprises,consists essentially of, or consists of, a liposome comprising a lipidbilayer having a hydrophilic exterior surface and an aqueous interiorand comprising at least one fusion polymer comprising (i) a tumortargeting domain that binds to exposed collagenous (XC) proteins, and(ii) a multi-block polymer comprising sequentially a first hydrophilicblock, a first hydrophobic block, a second hydrophobic block, and asecond hydrophilic block, wherein the tumor targeting domain of the atleast one fusion polymer extends outwardly from the hydrophilic exteriorsurface of the liposome, such that the tumor targeting domain can bindto XC proteins while associated with the liposome. In some embodiments,the liposome comprises, consists essentially of, or consists of, aplurality of the fusion polymers and wherein the fusion polymerscomprise at least or about 1/10^(th) (at least or about 2/10^(ths),3/10^(ths), 4/10^(ths), 5/10^(ths), 6/10^(ths), 7/10^(ths), 8/10^(ths)or at least or about 9/10^(th), e.g., 10/10^(ths), i.e., all) of thetotal number of polymers that comprise the liposome. In someembodiments, the tumor targeting aptamer comprises, consists essentiallyof, or consists of, SEQ ID NO: 1 or SEQ ID NO: 2 and in someembodiments, the tumor targeting domain binds to XC proteins present insolid tumors. In some embodiments, a pharmaceutical compositioncomprises, consists essentially of, or consists of, the fusionnanoparticle and a pharmaceutically acceptable carrier. Also includedare methods of treating cancer in a subject that include, e.g.,administering to the subject a pharmaceutically effective amount of thepharmaceutical composition described herein. The cancer can be a primaryor metastatic cancer as described herein.

In some embodiments, the drug-delivery or bifunctional fusionnanoparticle comprises, consists essentially of, or consists of, aliposome comprising at least or about one fusion polymer and at leastone drug sequestered inside the aqueous interior of the shell lipidbilayer of the liposome. In some embodiments the drug-delivery orbifunctional fusion nanoparticle comprises, consists essentially of, orconsists of, a liposome comprising at least or about one fusion polymerand at least one drug sequestered inside the lipid bilayer of theliposome. In some embodiments, a pharmaceutical composition comprises,consists essentially of, or consists of, the fusion nanoparticle and apharmaceutically acceptable carrier. Also included are methods oftreating cancer in a subject that include, e.g., administering to thesubject a pharmaceutically effective amount of the pharmaceuticalcomposition described herein. The cancer can be a primary or metastaticcancer as described herein.

In some embodiments, the drug sequestered inside the liposome comprises,consists essentially of, or consists of, a drug selected from the groupconsisting of: a hydrophobic drug, a hydrophilic drug, a nucleic acid, aTaxane, doxorubicin, epirubicin, a platinum drug, R547, acyclin-dependent kinase inhibitor, TGX-221, a PI-3 kinase inhibitor,captothecin, gemcitabine, 5-fluouracil, rifampicin, tamoxifen,ellipticin, ethotrexate, daunomycin, estrogen, curcumin, and an siRNA,or any combination thereof. In some embodiments, the drug is ahydrophobic drug sequestered in the hydrophobic layer of the bilayer. Insome embodiments, the drug is a hydrophilic drug encapsulated in theaqueous interior of the bilayer shell.

Also provided are methods of making a liposome comprising a lipidbilayer having a hydrophilic exterior surface and an aqueous interiorand comprising at least one fusion polymer comprising (i) a tumortargeting domain that binds to exposed collagenous (XC) proteins, and(ii) a multi-block polymer comprising sequentially a first hydrophilicblock, a first hydrophobic block, a second hydrophobic block, and asecond hydrophilic block, wherein the tumor targeting domain of the atleast one fusion polymer extends outwardly from the hydrophilic exteriorsurface of the liposome, such that the tumor targeting domain can bindto XC proteins while associated with the liposome. The method of makinga liposome can comprise (a) providing a first and a second plurality ofmulti-block polymers, each comprising sequentially a first hydrophilicblock, a first hydrophobic block, a second hydrophobic block, and asecond hydrophilic block, wherein the multi-block polymers of the secondplurality each comprise a linker bound to the terminus of the firsthydrophilic block, and wherein a maleimide group is bound to the linkerterminus that is unbound to the hydrophilic block; (b) mixing the firstand second plurality of multi-block polymers to thereby form a liposome;(c) providing a tumor-targeting domain that binds to exposed collagenousproteins and that comprises a cysteine residue on a terminus of thedomain; and (d) combining the liposome of (b) with the tumor-targetingdomain of (c) under conditions such that an activated maleimide of themulti-block polymers of the second plurality in the liposome reacts withthe cysteine residue of the tumor-targeting domain, thereby connectingthe tumor-targeting domain and the liposome.

In some embodiments the method of making a liposome comprises: (a)providing a first and a second plurality of multi-block polymers, eachcomprising sequentially a first hydrophilic block, a first hydrophobicblock, a second hydrophobic block, and a second hydrophilic block,wherein the multi-block polymers of the second plurality each comprise alinker bound to the terminus of the first hydrophilic block, and whereina maleimide group is bound to the linker terminus that is unbound to thehydrophilic block; (b) providing a tumor-targeting domain that binds toexposed collagenous proteins and that comprises a cysteine residue on aterminus of the domain; (c) combining the second plurality ofmulti-block polymers of (a) with the tumor-targeting domain of (b) underconditions such that an activated maleimide of the multi-block polymersof the second plurality in the liposome reacts with the cysteine residueof the tumor-targeting domain, thereby connecting the tumor-targetingdomain and the multi-block polymer and creating a fusion polymer; and(d) mixing the fusion polymer of (c) with the first plurality ofmulti-block polymers to thereby form a micelle.

In some embodiments, the method of making a liposome further comprises,during mixing, adding a therapeutic agent under conditions sufficient toform a liposome that encapsulates the therapeutic agent.

Also provided are methods of making a drug delivery nanoparticlecomprising (1) a liposome comprising a lipid bilayer having ahydrophilic exterior surface and an aqueous interior and comprising atleast one fusion polymer comprising (i) a tumor targeting domain thatbinds to exposed collagenous (XC) proteins, and (ii) a multi-blockpolymer comprising sequentially a first hydrophilic block, a firsthydrophobic block, a second hydrophobic block, and a second hydrophilicblock, wherein the tumor targeting domain of the at least one fusionpolymer extends outwardly from the hydrophilic exterior surface of theliposome, such that the tumor targeting domain can bind to XC proteinswhile associated with the liposome and (2) a drug sequestered inside theaqueous interior of the liposome. The method of making a drug deliverynanoparticle comprises (a) providing the drug; (b) providing theliposome; and (c) mixing the drug and the liposome to allow associationof the liposome with the drug, thereby forming a drug-deliverynanoparticle.

Provided herein are pharmaceutical compositions comprising any one ofthe fusion polymers described herein and a pharmaceutical acceptablecarrier. Also provided herein are kits that include one or more of thefusion polymers described herein.

Also provided are methods of treating cancer in a subject that include,e.g., administering to the subject a pharmaceutically effective amountof a pharmaceutical composition described herein. The cancer can be aprimary or metastatic cancer e.g., colorectal cancer, breast cancer,brain cancer, non-small cell lung cancer, pancreatic cancer, prostatecancer, a sarcoma, a carcinoma, or a melanoma. Also contemplated aretreatments of multiple types of cancer, simultaneously, using one ormore of the compositions described herein.

As used herein, the term “fusion protein” or “onco-aptamer” is apolypeptide containing portions of amino acid sequence derived from twoor more different proteins, or two or more regions of the same proteinthat are not normally contiguous.

The term “aptamer” is art-known and refers to an oligonucleotide orpolypeptide that binds to a specific target molecule with high affinityand specificity.

The terms “polypeptide” or “peptide” as used herein are intended toencompass any amino acid sequence and include modified sequences such asthose modified via PEGylation or acetylation. A “polypeptide” can berecombinantly or synthetically synthesized.

The term “micelle” or “polymer micelle” is art-known and means agenerally spherical nanoparticle structure of amphipathic molecules, ormolecules that have a polar head and a nonpolar tail, wherein thenonpolar tails are generally inside the micelle and shielded from waterby the polar heads that generally line the outside of the micelle.

The term “liposome” is art-known and means a nanoparticle made ofpolymers and having at least one closed membrane. The at least oneclosed membrane can comprise a single bilayer or may comprise two ormore concentrically arranged bilayers and the closed membrane can definean internal aqueous compartment.

The term “collagen-binding domain” is known in the art and refers to anypolypeptide, or portion thereof, that can bind collagen, e.g., thecollagenous proteins that are newly expressed and/or exposed in injuredtissues and invasive cancers, as well as collagenous affinity matricesprepared in vitro. Several collagen-binding domains are known in the art(Cruz M A, et al., J. Biol. Chem., 270:10822-10827, 1995; Hoylaerts, M.F., et al., Biochem. J., 324:185-191, 1997; Lankhof, H., et al.,Thrombos Haemostas, 75:950-958, 1996, which are incorporated herein intheir entirety).

The term “paclitaxel-binding domain” refers to any polypeptide, orportion thereof, that can bind paclitaxel. Several paclitaxel-bindingdomains have been described based on the suspected substrates thatpaclitaxel bind, including beta-tubulin (Rao et al., J. Biol. Chem.,1994, 269:3132-4; Rao et al., J. Biol. Chem., 1995, 270:20235-8; Amosand Lowe, Chem. & Biol. 1999, 6:R65-69); the anti-apoptotic proteinBc1-2 (Fang et al., Cancer Res., 1998, 89:3202-8; Rodi et al., J. Mol.Biol., 1999, 285:197-203); and the nuclear transcription factor NFX1(Aoki et al., Bioconjugate Chem., 2007, 18:1981-6; Katzenellenbogen etal., J. Virol., 2007, 81:3786-96, which are incorporated herein in theirentirety).

The term “immunoglobulin-binding domain” refers to any polypeptide, orportion thereof, that can bind immunoglobulin.

As used herein, the term “monoclonal antibody” refers to a population ofantibody molecules that include only one species of an antigen bindingsite capable of immune-reacting with a particular epitope of apolypeptide or protein. A monoclonal antibody thus typically displays asingle binding affinity for the protein to which it specifically binds.

The term “RNA-binding domain” refers to any polypeptide, or portionthereof, that can bind ribonucleic acid (RNA).

The term “human serum albumin-binding domain” or “HSA-binding domain”refers to a structure that comprises a thiol-reactive maleimide group ora polypeptide, or portion thereof, that can bind human serum albumin.

The term “treatment” refers to the administration of one or morepharmaceutical agents to a subject or the performance of a medicalprocedure on the body of a subject. The term treatment also includes anadjustment (e.g., increase or decrease) in the dose or frequency of oneor more pharmaceutical agents that a subject can be taking, theadministration of one or more new pharmaceutical agents to the subject,or the removal of one or more pharmaceutical agents from the subject'streatment plan.

As used herein, a “subject” is an animal, e.g., a mammal. A specificexample of a subject would be, e.g., a human, e.g., an adult human orjuvenile. Veterinary applications are also contemplated and a subjectcan be, e.g., a monkey, dog, cat, horse, cow, pig, goat, rabbit, rat, ormouse.

An “effective amount” is an amount sufficient to effect beneficial ordesired results. For example, a therapeutically effective amount is onethat achieves the desired therapeutic effect. An effective amount can beadministered in one or more administrations, applications or dosages. Atherapeutically effective amount of a pharmaceutical composition (i.e.,an effective dosage) depends on the pharmaceutical composition selected.The compositions can be administered from one or more times per day toone or more times per week; including once every other day. The skilledartisan will appreciate that certain factors may influence the dosageand timing required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of the pharmaceutical compositions described herein caninclude a single treatment or a series of treatments.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1A is a diagram illustrating the histology of normal tissue andshows that in normal, uninjured tissues, collagen is not exposed to thegeneral circulation.

FIG. 1B is a diagram illustrating the histology of tumor tissue andshows that tumor invasion, metastasis, angiogenesis, and reactive stromaformation result in the disruption of the normal histology and thepathologic exposure of collagenous XC-proteins within the tumormicroenvironment.

FIG. 2A is a diagram illustrating the two-handed structure of thebifunctional fusion polymers. The fusion polymers contain two primarybinding elements: (1) a XC-binding domain (tumor-targeting domain) and(2) a drug-binding domain.

FIG. 2B is a diagram illustrating the molecular design of arepresentative fusion polymer (onco-aptamer) with a XC-bindingtumor-targeting domain and a drug binding domain.

FIG. 2C is a diagram illustrating the two-handed structure of thebifunctional fusion polymers. The fusion polymer demonstrated herecontains two primary elements: (1) and XC-binding domain(tumor-targeting domain) and (2) a drug-binding domain that can bindIgGs.

FIG. 2D is a diagram illustrating targeted drug delivery using HumanSerum Albumin (HSA)-aptamer fusions. In this tumor-targeting HSA-bindingsystem, many therapeutic agents bind tightly to albumin, and thisinteraction can increase the half-life of the therapeutic agent incirculation.

FIG. 2E is a diagram illustrating different embodiments of XC-bindingHSA binding fusion polymers used for the tumor-targeted delivery oftherapeutic agents bound to HSA.

FIG. 3A is a diagram that illustrates the FITC-labeled collagen-bindingconstructs.

FIG. 3B is an image that shows the collagen-agarose chromatographyresults of selected FITC-labeled collagen-binding constructs.

FIG. 3C is a nondestructive image that shows fluorescence (arrow) of asubcutaneous tumor in a nude mice intravenously injected with aFITC-labeled collagen-binding construct.

FIG. 3D is an image showing fluorescence of a subcutaneous tumor from anude mice intravenously injected with a FITC-labeled collagen-bindingconstruct.

FIG. 4A is a diagram showing the structures of three Taxol-Tropinaptamers (Tx-Aptamers).

FIG. 4B is an image that shows the collagen-agarose columnchromatography results of these Tx-Aptamers.

FIG. 4C is an image that shows the comparative serum stability(resistance to proteolysis) of these Tx-Aptamerss.

FIG. 5A is an image that shows that MDA-MB-231 breast cancer cells onControl collagen beads readily adhere and proliferate (dashed circles),spreading out along the XC surfaces of the bead matrix at 48 and 72hours.

FIG. 5B is an image that shows that the fusion XC-binding/Taxol-bindingcomplexes are biologically active: MDA-MB-231 breast cancer cells onTaxol-Aptamer beads showed the classical signs of cell death anddestruction, that is, a mitotic block followed by apoptosis, necrosis,and cytolysis (arrows).

FIG. 5C is an image that shows collagen-agarose column chromatography ofcontrol and Taxol-Aptamers, which were used in the cell cultures.

FIG. 6A is a diagram illustrating targeting of paclitaxel byTaxol-Aptamers to subcutaneous tumors in nude mice and an image of thesubcutaneous tumor in a nude mouse.

FIG. 6B is a panel of images that show Taxol-aptamer dependent tumortargeting of paclitaxel, shown here by the comparative fluorescence ofexcised tumors.

FIG. 6C is an image that shows that when used with Taxol-aptamer,paclitaxel exhibited anti-angiogenic activity (i.e., potential efficacy)at 0.2 mg/kg, only 1/50th of the normal effective dose of paclitaxelused in mice (10 mg/kg).

FIG. 7A is a diagram showing the structures of four mAb-Binding,collagen-binding fusion peptides that were generated and tested.

FIG. 7B is an image that shows the collagen-agarose columnchromatography results of the four mAb-aptamers from FIG. 7A.

FIG. 7C is an image that shows the serum stability (resistance toproteolysis) of mAb-Aptamer 1, mAb-Aptamer 2 and mAb-Aptamer 4.

FIG. 8A is a diagram showing the structures of two representativemAb-Tropins (M03 and P38).

FIG. 8B is an image that shows that the combined antibody-binding andcollagen-binding activity of M03 is positive and peptide-dependent,while that of P38 is nearly negative.

FIG. 8C is a graph that shows that increasing the concentrations of theM03 mAb-Aptamer also increased the collagen-bound retentates of Avastin(a humanized mAb) in the XC-agarose columns.

FIG. 8D is an image that shows that increasing the concentrations of theM03 mAb-Aptamer also increased the collagen-bound retentates of Avastin(a humanized mAb) in the XC-agarose columns.

FIG. 9A is an image of VEGF-stimulated growth and proliferation of HUVECcells in control columns (control medium eluate). Mitotic cells areidentified by white arrows.

FIG. 9B is an image of VEGF-stimulated growth and proliferation of HUVECcells in control columns (control medium eluate). Mitotic cells areidentified by white arrows.

FIG. 9C is an image of VEGF-stimulated growth and proliferation of HUVECcells in control columns (control medium eluate). Mitotic cells areidentified by white arrows.

FIG. 9D is an image illustrating that the VEGF-stimulated growth andproliferation of the HUVEC cells was blocked in VEGF-Trap(collagen/mAb-Tropin/Avastin complex) columns, demonstrating that thecollagen-bound Aptamer-Avastin complexes retained their biologicalactivity.

FIG. 9E is an image illustrating that the VEGF-stimulated growth andproliferation of the HUVEC cells was blocked in VEGF-Trap(collagen/mAb-Tropin/Avastin complex) columns, demonstrating that thecollagen-bound Aptamer-Avastin complexes retained their biologicalactivity.

FIG. 9F is an image illustrating that the VEGF-stimulated growth andproliferation of the HUVEC cells was blocked in VEGF-Trap(collagen/mAb-Tropin/Avastin complex) columns, demonstrating that thecollagen-bound Aptamer-Avastin complexes retained their biologicalactivity.

FIG. 10A is a diagram showing the structures of two RNA-binding andcollagen-binding fusion peptides (RNA-Apt 07 and RNA-Apt 08) that weregenerated and tested.

FIG. 10B is an image that shows that both RNA-Apt 07 and RNA-Apt 08depleted fluorescently labeled-oligonucleotides from the column eluates.

FIG. 10C is an image that shows that both RNA-Apt 07 and RNA-Apt 08retained the labeled oligonucleotides on the collagen-agarosemini-columns.

FIG. 11A is a panel of bright field and fluorescence images of a tumorexcised from a mouse treated with intravenous (tail vein) infusions ofIgG^(FITC) minus the collagen-binding/IgG-binding fusion aptamer.

FIG. 11B is a panel of bright field and fluorescence images of a tumorexcised from a mouse treated with intravenous (tail vein) infusions ofIgG^(FITC) plus the collagen-binding/IgG-binding fusion aptamer,demonstrating XC-binding domain-dependent tumor targeting of IgGs.

FIG. 11C is a panel of bright field and fluorescence images of a controltumor (untreated), used to determine the level of backgroundauto-fluorescence.

FIG. 12 is an image illustrating a functional analysis, via comparativeXC-column chromatography, of the maleimide-activated binding ofmulti-human serum albumin (HSA) tropins (Aptamer-3 and Aptamer-4) to thefree cysteine-34 of recombinant HSA. The collagen-binding/albumin fusionpolymers were detected using FITC-labeled goat anti-HSA antibodies.

FIG. 13 is a schematic of the novel tripartite onco-aptamer,CpBio-MA3TX, named TargaTaxel. This schematic shows the (i)maleimide-activated Albumin linkage; (ii) a provencollagen-binding/tumor-targeting domain; and (iii) a high efficiencyTaxol-binding domain.

FIG. 14A is a schematic of an exemplary tumor-targeting bifunctionalfusion nanoparticle system, illustrating the structure of amaleimide-activated di-block copolymer and a schematic of a bifunctionalpolymer formed by the linking of a tumor-targeted collagen-bindingonco-aptamer and a maleimide-activated di-block copolymer.

FIG. 14B is a schematic of an exemplary bifunctional fusion nanoparticletumor-targeting system, illustrating a drug loaded nano-micelle.

FIG. 14C is a schematic of an exemplary bifunctional fusion nanoparticletumor-targeting system, illustrating a drug delivery nanoparticlecreated with tumor-targeted polymers of the present invention.

FIG. 15A is a schematic of two tumor-targeted collagen-binding peptides.

FIG. 15B is an image of an XC-agarose chromatography analysis ofpolymeric micelles loaded with Courmarin-6 and coupled to thetumor-targeting peptides from FIG. 15A.

FIG. 15C is a graph of the quantification of the column eluates of FIG.15B, confirming that Aptamer 1 (N-terminal Cys) demonstrates greatercoupling efficiency.

FIG. 16A is a bright field image illustrating selectivity of aTaxol-Tropin fusion polymer for the XC proteins of a collagen-agarosematrix. The aptamer-dependent (Tx-Aptamer 2 from FIG. 4A) binding offluorescent paclitaxel (Taxol-green) to the XC-agarose layer isdemonstrated by column chromatography.

FIG. 16B is a blue-light/amber filter trans-illumination imageillustrating selectivity of a Taxol-Tropin fusion polymer for the XCproteins of a collagen-agarose matrix. The aptamer-dependent (Tx-Aptamer2 from FIG. 4A) binding of fluorescent paclitaxel (Taxol-green) to theXC-agarose layer is demonstrated by column chromatography.

DETAILED DESCRIPTION

The present disclosure is based, at least in part, on the development ofnew bifunctional fusion polymers that include at least two functionaldomains: (i) sequences that bind to the Exposed Collagenous (XC-)proteins present in pathological areas such as cancerous lesions, and(ii) sequences that bind directly or indirectly to a particular class ofchemotherapeutic or biologic agents, for example, paclitaxel, monoclonalantibodies, growth factors, or small interfering RNA (siRNA). Thepresent disclosure is also based, at least in part, on the developmentof new bifunctional fusion nanoparticles that include at least twofunctional portions: (i) a least one fusion polymer that includes asequence that binds to Exposed Collagenous (XC-) proteins, and anamphiphilic polymer with distinct hydrophobic and hydrophilic blockdomains that self-assemble with similar polymers to assemble acore-shell structure, and (ii) a nanoparticle that non-covalentlysequesters chemotherapeutic or biologic agents, for example agents withhydrophobic or hydrophilic characteristics or nucleic acids. Thebifunctional properties of these engineered fusion polymers andnanoparticles may enable selective and efficient targeting of the widelyused chemotherapeutic and biologic agents to abnormal, diseased, ordegenerative tissues such as tumors, allowing lower doses of theseagents to become more effective at killing cancer cells and associatedblood supply. Targeting is achieved by combining a tumor-targetingfunctional domain with a high-affinity, non-covalent drug-binding domainof the fusion polymers or with drug-delivery nanoparticle, generatingdrug complexes with improved biodistribution. Targeted delivery of drugsusing the fusion peptide and nanoparticles disclosed herein can reducesystemic toxicity and side effects by sequestering the drugs in thetumor microenvironment and sparing normal cells and tissues from thetoxicity of the drugs. Moreover, by targeting a common histopathologicproperty of primary tumors and metastatic lesions, the drug deliverysystems described herein can (i) bind and carry one or more FDA-approvedtherapeutic drugs, in some instances upon simple mixing, and (ii) seekout and accumulate in the diseased/cancerous tissues followingintravenous infusion. Thus, the fusion polymers and nanoparticlesdescribed herein may make conventional chemotherapeutic and biologicagents more efficient with great efficacy while lessening unwanted sideeffects, improving the overall Therapeutic Index and patient survival.These fusion polymers and nanoparticles can also include linker segmentsand/or flanking sequences to improve the functionality,pharmacokinetics, stability, and/or pharmacodynamics of the targeteddrug delivery.

Selective Targeting of Therapeutic Agents to Tumor Microenvironment

Neoplastic lesions do not only comprise malignant cancer cells but alsoinclude stromal components such as fibroblasts, endothelial cells, andinflammatory cells. An opportunistic tumor microenvironment is formed bythose components and promotes tumorigenesis, tumor progression andmetastasis. Although cancer drug development traditionally focused ontargeting the cancer cell and its cell division cycle, emphasis hasrecently shifted toward the tumor microenvironment for novel therapeuticand prevention strategies (See Sounni and Noel, Clinical Chem.,59:85-93, 2013; Fang and DeClerck, Cancer Res., 73:4965-4977, 2013). Asshown in FIG. 1, the process of tumor invasion, metastasis,angiogenesis, and reactive stroma formation disrupts normal tissuehistology and leads to pathologic exposure of collagenous proteins (XC-)within the tumor microenvironment. Thus, the abnormal exposure ofcollagenous proteins is a characteristic histopathologic property of allneoplastic lesions.

“Pathotropic (disease-seeking) Targeting” of drugs to cancerous tissuesutilizes the pathology of tumor itself (XC-protein expression) as thebiochemical target, rather than the unique, varied, rapidly evolvingcancer cells per se. By targeting a common histopathologic property ofprimary tumors and metastatic lesions, the drug delivery systemsdescribed herein can (i) bind and carry a well-characterized (e.g.,FDA-approved) therapeutic drug, upon simple mixing, and (ii) seek-outand accumulate in the diseased/cancerous tissues upon intravenousinfusion. The advent of Pathotropic Targeting ushered the field ofgenetic medicine into the clinic (Waehler et al., Nature ReviewsGenetics 8:573-587, 2007). Further advancement of the field of targetedantitumor therapy was made with the development of Rexin-G (Hall et al.,Hum Gene Ther 11:983-993, 2000; Gordon et al., Cancer Res. 60:3343-3347,2000; Hall et al., Intl J Mol Med 6:635-643, 2000)—a nanoparticle genedelivery system which incorporates a physiological surveillance functioninherent in the primary structure of von Willebrand Factor—to enable aspecific gain-of-function that is highly selective for the pathologicstroma that is characteristic of neoplastic lesions (Gordon et al.,Cancer Res. 60:3343-3347, 2000). Indeed, the clinical administration ofthis tumor-targeted Rexin-G vector has been shown to accumulate inprimary and metastatic lesions, resulting in enhanced cytotoxic genedelivery, and thus enhanced clinical efficacy (Gordon et al., ExpertOpin Biol Ther 10:819-832, 2010; Gordon et al., Int'l J Oncol36:1341-1353, 2010; Chawla et al., Mol Ther 2009; 17(9):1651-7; Chawlaet al., Mol Ther 2010; 18:435-441).

To avoid the problems caused by traditional chemotherapeutic regimens, amodality of drug administration called “metronomic chemotherapy” hasbeen proposed, which refers to the chronic, equally spacedadministration of low doses of various chemotherapeutic drugs withoutextended rest periods. Metronomic chemotherapy targets endothelial cellsrather than tumor cells.

Provided herein are novel bifunctional fusion polymers (also called“onco-aptamers”) that target pharmaceutical agents to pathologicalareas. The fusion polymers described herein contain two or more domains:(i) sequences that bind to Exposed Collagenous (XC-) proteins present inpathological areas, including cancerous lesions, and (ii) sequences thatbind to specific pharmaceutical agents (see FIG. 2A). These fusionpolymers can also include (iii) linker segments and/or (iv) flankingsequences to improve the functionality, pharmacokinetics, stability,and/or pharmacodynamics of the targeted drug delivery (see FIG. 2B).Also provided herein are novel bifunctional fusion nanoparticles fortargeting pharmaceutical agents to pathological areas. The fusionnanoparticles can include two or more portions: (i) at least one fusionpolymer that includes a sequence that binds to Exposed Collagenous (XC-)proteins present in pathological areas, such as cancerous lesions, andan amphiphilic polymer with distinct hydrophobic and hydrophilic blockdomains that self-assemble with similar polymers to assemble acore-shell structure, and (ii) a nanoparticle that covalently sequesterschemotherapeutic or biologic agents (e.g., FIG. 14A). These fusionpolymers can also include (iii) linker segments and/or (iv) flankingsequences to improve the functionality, pharmacokinetics, stability,and/or pharmacodynamics of the targeted drug delivery (e.g., FIG. 14A).

The molecular engineering of these bifunctional polymers andnanoparticles creates a two-handed structure with at least two distinctbinding domains separated by flexible linkers or spacers—to avoid sterichindrances between the functional domains. These fusion polymers andnanoparticles enable tumor-targeted delivery of the chemotherapeutic andbiologic agents, e.g., anticancer agents.

The drug-binding properties of these fusion polymers and nanoparticlesare based on high-affinity, non-covalent interactions, which do notsubstantially alter the chemical composition or the commercialmanufacturing of the pharmaceutical agents. The complete elimination ofthe bioactive therapeutic agent from the primary structure of thesetargeting fusion polymers greatly reduces the overall size of theconstructs to less than 50 amino acids (˜5 kDa), which is therebyamenable to GMP production by chemical synthesis, avoiding theproduction and purification methodologies required for larger proteinsprepared from biologic sources that are associated with a myriad ofmedical, pharmacological, and regulatory concerns. The chemicalstructure and pharmaceutical purity of these newly-developed tumortargeting fusion polymers and nanoparticles are readily verifiable—byvirtue of the synthetic chemistries involved. Furthermore, the chemicalcomposition and commercial manufacture of the bioactive pharmaceuticalagents to be delivered by these targeting peptides and nanoparticles arenot substantially altered due to the non-covalent binding.

The fusion polymers and nanoparticles described herein have acollagen-binding domain. In some embodiments, the collagen-bindingdomain is derived from a collagen-binding domain of von Willebrandfactor, which is involved in the recognition of exposed vascularcollagen (Takagi, J., et al., Biochemistry 32:8530-4, 1992; Tuan, T. L.,et al., Conn. Tiss. Res. 34:1-9, 1996; Gordon, E. M., et al., Hum. GeneTher. 8:1385-1394; U.S. Pat. No. 6,387,663, all herein incorporated byreference). von Willebrand factor was initially identified as ahemostatic factor in studies of inherited hemophilias (Wagner, Ann. Rev.Cell. Biol. 6:217, 1990), and has been shown to perform a vitalsurveillance function by targeting platelet aggregates toinjured/diseased tissues and vascular lesions (Ginsburg and Bowie, Blood79:2507-2519, 1992).

In some embodiments, the collagen-binding domain comprises an amino acidsequence of SEQ ID NO: 1 or SEQ ID NO: 2 or both. Skilled practitionerswill appreciate that variations in these sequences are possible and suchvariants may be useful in the present invention. Accordingly, sequenceshaving at least 70%, e.g., at least 80%, 90%, or at least 99% identityto SEQ ID NO.: 1 or SEQ ID NO.: 2 can be utilized.

Tumor-Targeting and Paclitaxel-Binding Fusion Polymers (Taxol-Tropins)

The taxanes are a group of drugs that includes paclitaxel (Taxol®) anddocetaxel (Taxotere®) that exhibit antitumor activity against a widerange of human cancers by binding to and stabilizing microtubules. Sincemicrotubules are essential to cell division, taxane-binding andstabilization of microtubules inhibits cell division and disrupts cellcycle. The efficacy of taxane-based therapy is often limited by systemictoxicity, which results in poor therapeutic index. The systemic toxicityof taxanes is due to their non-selective cytotoxicity toward tumor cellsversus normal cells. This indiscriminate property results in severe sideeffects, including bone marrow suppression, febrile neutropenia,neurotoxicity, mucositis, ulceration of the mouth and throat, as well asa variety of cardiac abnormalities. This untoward toxicity oftaxane-based therapy has restricted the administration and dose levels,which often lead to incomplete tumor eradication.

One way to reduce the side effects of taxanes is to directly targettaxanes to the primary and metastatic tumor sites. Such selective tumortargeting can increase the bioavailability of the drug within thetumors. Albumin drug complexes can reach tumors passively to some extentthrough the leaky vasculature surrounding the tumors by the EnhancedPermeability and Retention (EPR) effect. However, recent studiesperformed in a directly comparative manner have had a sobering effect:the results of a recent NCI-sponsored Phase III breast cancer trialcomparing paclitaxel with albumin-based nab-paclitaxel (Abraxane)determined that i.v. paclitaxel (Taxol) performed just as good or betterthan Abraxane, with significantly less toxicity (Rugo et al., 2015Randomized Phase III Trial of Paclitaxel Once Per Week Compared WithNanoparticle Albumin—Bound Nab—Paclitaxel Once Per Week or IxabepiloneWith Bevacizumab as First-Line Chemotherapy for Locally Recurrent orMetastatic Breast Cancer: CALGB 40502/NCCTG N063H (Alliance)., J.Clinical Oncology, 33:2361-2369). Drugs such as Taxol and Abraxane(which are administered at necessarily high (equi-toxic) doses) could befurther improved by active targeting. The use of active targetingtechnology could enable lower (not higher) doses of taxanes to becomemore clinically effective.

In some embodiments, the fusion polymers described herein comprise acollagen-binding domain and a paclitaxel-binding domain. These fusionpolymers can directly bind to paclitaxel and target paclitaxel toneoplastic lesions. In some embodiments, the paclitaxel-binding domainof the fusion polymers is derived from a paclitaxel-binding domain ofNFX1. For example, the paclitaxel-binding domain can comprise an aminoacid sequence selected from the group consisting of: SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. Skilled practitioners willappreciate that variations in such sequences may be possible, whileretaining paclitaxel-binding activity. Accordingly, sequences having atleast 70%, e.g., at least 80%, 90%, 95%, or at least 99%, identity toSEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, can beutilized.

In some embodiments, the tumor-targeting and paclitaxel-binding fusionpolymers further include one or more linkers/spacers. Theselinkers/spacers can add flexibility, reducing the steric hindrancesbetween the functional binding domains. The tumor-targeting andpaclitaxel-binding fusion polymers can have one or more linkerscomprising an amino acid sequence such as SEQ ID NO: 7, SEQ ID NO: 8,SEQ ID NO: 9, or SEQ ID NO: 10, or variants of these sequences. In someembodiments, the fusion polymer can be acetylated, amidated, and/orPEGylated at N- or C-terminus. Alternatively or in addition, D-aminoacids can be included. Inclusion of D-amino acids, acetylation,amidation, and/or PEGylation increase the stability of the fusionpolymers and make them more resistant to proteolysis.

Exemplary amino acid sequences of tumor-targeting and paclitaxel-bindingfusion polymers include SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 andSEQ ID NO: 14. Skilled practitioners will appreciate that variations insuch sequences may be possible, while retaining tumor- andpaclitaxel-binding activity. Accordingly, sequences having at least 70%,e.g., at least 80%, 90%, 95%, or at least 99%, identity to SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, can be utilized.

Tumor-Targeting and Monoclonal Antibody (mAb)-Binding Fusion Polymers(mAb-Tropins)

The use of monoclonal antibodies (mAbs) for cancer therapy has achievedconsiderable success in recent years. In a relatively short period oftime, mAbs have entered the mainstream of anticancer therapy. Monoclonalantibodies were first used as antagonists of oncogenic receptor tyrosinekinases, but today mAbs have emerged both as vehicles for the targeteddelivery of potent chemotherapeutic agents and as powerful tools tomanipulate tumor angiogenesis and anti-tumor immune responses. With evermore promising results from the clinic, the future will likely seecontinued growth in the discovery and development of therapeuticantibodies.

In some embodiments, the fusion polymers described herein comprise acollagen-binding domain and an immunoglobulin-binding domain (FIG. 2A).These fusion polymers can directly bind to immunoglobulins such asmonoclonal antibodies and target them to neoplastic lesions. A minimalIgG-binding domain of 35 amino-acids, corresponding to an N-terminaldomain of the fibrinogen-binding protein FgBP, was previously reported(Meehan et al., Microbiology, 2001, 147:3311-22; 2009, 155:2583-92). Thetethering of mAbs to a synthetic fragment of the deduced IgG-bindingdomain of FgBP, as well as IgG-binding peptide sequences determined bybio-panning (DeLano et al., 2000, Science, 287:1279-83; Yang et al., J.Peptide Res., 66: 120-137; 2005), are amenable to target mAb to tumors.

In some embodiments, the immunoglobulin-binding domain of the fusionpolymers comprises the hexamer sequence of SEQ ID NO: 15, which has beenshown to bind to the Fc region of human, bovine, mouse, goat, and rabbitimmunoglobulins (IgGs)—(Yang et al., J. Chromatography, 2009,1216:910-18; Yang et al., J. Mol. Recognition, 2010, 23:271-82, whichare incorporated herein in their entirety). Skilled practitioners willappreciate that the sequence of SEQ ID NO:15 could be modified andtested to modify or retain at least some portion of its Fc bindingactivity and, accordingly, variants (i.e., sequences with at least 70%,e.g., at least 80% or at least 90% identity to SEQ ID NO:15) can beuseful in certain embodiments.

In some embodiments, the tumor-targeting and immunoglobulin-bindingfusion polymers further include one or more linkers/spacers, to reducethe steric hindrances between the functional binding domains. Thetumor-targeting and immunoglobulin-binding fusion polymers can have oneor more linkers comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 57, and SEQ ID NO: 58, or variants of these sequences.In some embodiments, the fusion polymers can be acetylated, amidated,and/or PEGylated at N- or C-terminus. Alternatively or in addition,D-amino acids can be included. Inclusion of D-amino acids, acetylation,amidation, and/or PEGylation can increase the stability of the fusionpolymers and can make them more resistant to proteolysis.

Exemplary amino acid sequences of tumor-targeting andimmunoglobulin-binding fusion polymers include SEQ ID NO: 18, SEQ ID NO:19, SEQ ID NO: 20 and SEQ ID NO: 21. Skilled practitioners willappreciate that variations in such sequences may be possible, whileretaining tumor- and immunoglobulin-binding activity. Accordingly,sequences having at least 70%, e.g., at least 80%, 90%, 95%, or at least99%, identity to SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ IDNO: 21, can be utilized.

In some embodiments, the immunoglobulin-binding domain described hereincan bind monoclonal antibodies such as immune checkpoint inhibitors,thereby serving to restrict antibody-mediated immune activation to tumorcompartments. Exemplary immune or T-cell checkpoint inhibitors includeanti-cytotoxic T lymphocyte antigen-4 (CTLA-4), anti-programmed death-1(anti-PD-1) and anti-PD-ligand-1 (anti-PD-L1) monoclonal antibodies. Theuse of T-cell checkpoint inhibitors for breaking immune tolerance is amajor advancement in cancer immunology. Under normal physiologicalconditions PD-L1 binds to the transmembrane PD-1 on an immune cellsurface and inhibits the immune activity of the cell. Cancer cells canupregulate PD-L1 so as to inhibit the T-cells that might otherwiseattack the tumor cells. Antibodies such as anti-PD-1 mAbs block theinteraction between PD-L1 and PD-1, activating the immune system of theT-cell so that it can attack the tumor. Similarly CTLA-4 is an antibodyexpressed on the surface of T-cells that transmits inhibitory signal inthe T-cell. This inhibitory signal can be blocked with anti-CTLA-4antibodies, such that the immune system can recognize and target cancercells.

The use of anti-CTLA-4, anti-PD-1 or PD-L1 antibodies is associated witha wide range of side effects known as immune-related adverse events(irAEs), which can impact dermatologic, gastrointestinal, hepatic,endocrine, and other organ systems, requiring subsequentimmunosuppression with corticosteroids, tumor necrosis factor-alphaantagonists, mycophenolate mofetil, or other agents (Kong and Flynn,2014; Chen et al., 2015; Postow et al., 2015). Thus, the therapeuticperformance of such monoclonal antibodies can be enhanced by activetumor-targeting: thereby increasing the efficacy of the treatmentswithin the tumor compartments while decreasing the autoimmune disordersand untoward inflammatory sequelae caused by the bioactivity of thesecheckpoint inhibitors in non-target organs.

These immune checkpoint inhibiting fusion polymers can comprise the fulllength mAb or a functional fragment thereof, and can further include thelinkers/spacers and N-terminal/C-terminal shielding as described herein.Accordingly, included herein are methods of treating a disease, forexample, cancer. The cancer can be, e.g., a primary or metastaticcancer, including but not restricted to, colorectal cancer, breastcancer, brain tumors, non-small cell lung cancer, pancreatic cancer,prostate cancer, sarcoma, carcinoma, and/or melanoma. The cancer can be,e.g., cancer of the stomach, colon, rectum, mouth/pharynx, esophagus,larynx, liver, pancreas, lung, breast, cervix uteri, corpus uteri,ovary, prostate, testis, bladder, skin, bone, kidney, brain/centralnervous system, head, neck and/or throat; sarcoma, or choriocarcinoma.In general, the methods of treating cancer can include administering toa subject having cancer an amount of the immune-checkpoint inhibitingfusion polymer or pharmaceutical composition sufficient to treat cancerin the patient. The use of a tumor-targeted XC polymer fused with immunecheckpoint inhibitors can compartmentalize the T-cell response in thetumor microenvironment (TME), increase the efficacy of T cell responselocally in the TME, and reduce the severity of systemic immune-mediatedadverse events.

Tumor-Targeting and RNA-Binding Fusion Polymers (RNA-Tropins)

RNA interference (RNAi) using small interfering RNA sequences (siRNA) isone of the well-established strategies for gene silencing and cancertherapy (Burnett and Rossi, Cell, 19:60-71, 2012; Esposito et al., J.RNA Silencing, 10:500-506, 2014). To utilize RNAi for gene therapyapplications, however, an efficient and safe method for delivering thetherapeutic RNA molecules to the diseased tissues is both necessary andchallenging (Bae and Park, J. Controlled Release, 153:198-205, 2011;Ling et al., Nature Reviews Drug Discovery, 12:847-855, 2013). Toimprove targeting specificity, the RNA delivery vehicles and/or vectorsmust be functionalized or chemically modified (Copolovici et al., ACSNano, 8:1972-1994, 2014). In terms of cellular delivery andinternalization, the plasma membrane is the primary, and most difficult,barrier for RNA to cross, because negatively charged RNA is stronglyrepulsed by the negatively charged cellular membrane. A variety ofcationic polymers can be used as RNA carriers by interacting with RNAand covering its negative charges to form a cell-penetratingnanoparticle complex (Munyendo et al., Biomolecules, 2:187-202, 2012).Cationic cell-penetrating peptides (CPPs) are promising candidates forRNA carriers since they can cross the plasma membrane and beinternalized into cells together with the “cargo” RNA (Bechara andSagan, FEBS Letters, 587:1693-1702, 2013).

CPPs are short chains of amino acids with the distinct ability to crosscell plasma membranes. They are usually between seven and thirtyresidues in length. The commonality between all known CPPs is thepresence of positively charged residues within the amino acid chain.Polyarginine and the transactivator of transcription (TAT) peptide aretwo widely used CPPs (Futaki et al., J. Biol. Chem. 2001, 276:5836-5840;Schmidt et al., FEBS Letters, 2010, 584:1806-13). One advantage of CPPsis the ability to enhance the therapeutic delivery of a wide range oflarge-cargo molecules, such as oligonucleotides, into target cells(Zorko and Langel, Adv. Drug Delivery Rev., 57:529-545, 2004).

In some embodiments, the fusion polymers described herein comprise acollagen-binding domain and a RNA-binding domain. These fusion peptidescan directly bind to siRNA and target them to neoplastic lesions. Insome embodiments, the RNA-binding domain of the fusion polymerscomprises CPPs such as polyarginine or the TAT peptide. In someembodiments, the RNA-binding domain comprises polyarginine SEQ ID NO: 29or SEQ ID NO: 30.

In some embodiments, the tumor-targeting and RNA-binding fusion polymersfurther include one or more linkers/spacers, to reduce the sterichindrances between the functional binding domains. The tumor-targetingand RNA-binding fusion polymers can have one or more linkers comprisingan amino acid sequence selected from the group consisting of SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO: 8, SEQ ID NO: 58, SEQ ID NO: 33 and SEQ IDNO: 34, or variants of these sequences. In some embodiments, the fusionpolymers are amidated, acetylated and/or PEGylated at N- or C-terminus.Inclusion of D-amino acids, amidation, acetylation, and/or PEGylationincrease the stability of the fusion polymers and make them moreresistant to proteolysis.

Exemplary amino acid sequences of tumor-targeting and RNA-binding fusionpolymers include: SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ IDNO: 38, and SEQ ID NO: 55. Skilled practitioners will appreciate thatvariations in such sequences may be possible, while retaining tumor- andRNA-binding activity. Accordingly, sequences having at least 70%, e.g.,at least 80%, 90%, 95%, or at least 99%, identity to SEQ ID NO: 35, SEQID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO.: 55 can beutilized.

Tumor-Targeting and Albumin-Binding Fusion Polymers (Multi-Tropins)

Albumin, a versatile protein carrier for drug delivery, has been shownto be nontoxic, non-immunogenic, biocompatible, and biodegradable.Albumin-mediated drug delivery systems have gained considerableattention owing to their high binding capacity of various drugs, and thetendency of good tolerance and less side-effects. Albumin was used tofabricate various nanoparticles and targeting vehicles for improving thetherapeutic delivery of many drugs (Kratz, J. Controlled Release, 2008,132:171-83; Kinam Park, J. Controlled. Release, 2012, 157:3; Kwon etal., J. Controlled Release, 2012, 164:108-14). Human serum albumin (HSA)can bind and transport copper (Quinlan et al., Hepatology, 41:1211-1219,2005), including copper-based radiopharmaceuticals and PET imagingtracers (Lux et al., Theranostics, 5:277-288, 2015).

In 1986, Hiroshi Maeda intravenously injected the albumin-binding dyeEvans Blue into mice bearing subcutaneous tumors and found that theEvans blue-albumin complexes accumulated discernibly within the tumors.This phenomenon of enhanced permeability and retention—in relation topassive tumor targeting—is largely a result of the abnormal endothelialcell organization and the large fenestrations of tumor vasculature thatmake the tumor tissue more permeable for albumin. Due to its passiveentry into tumors via the enhanced permeability of tumor vasculature anda retention effect caused by increased demand for albumin by tumor cellsas a source of energy and amino acids, albumin-based drug deliverysystems have been shown to be useful for achieving improved cancerchemotherapy. The delivery of insoluble taxanes, for example, has beenimproved by the use of lipid-based solvents and albumin as a vehicle.The most popular formulation of albumin-bound paclitaxel (i.e.,Abraxane) facilitates the infusion of this biocompatible vehicle,improves the tolerability, and lessens side effects; although enhancedefficacy is generally seen with higher, not lower, drug doses (Miele etal., Int. Journal of Nanomedicine, 2009, 4:91-97). Moreover, the U.S.FDA has made it clear that the passive tumor targeting by albumin doesnot provide the selectivity required for specific tumor-targetingalthough it may improve the solubility and/or the clinical dosing ofdrugs (Department of Health & Human Services, NDA #021660 Abraxane® forInjectable Suspension. Reference ID: 3063889).

The major problem with the passive targeting of albumin-drug complexesto tumors is the fact that there is upwards of 35 to 50 grams of normalserum albumin in a single liter of human blood—that amounts to 175 to250 grams—which effectively competes with the milligram quantities ofthe drug and albumin components of these simplistic formulations.Therefore, active targeting must, necessarily, be several orders ofmagnitude higher in the affinity for tumor constituents to be trulyeffective. Integrin-binding Cyclic RGD peptides and their derivativeshave been intensively studied as highly selective tumor targetingprobes; however, the rather short blood circulation half-lives, greatlycompromises their targeting efficacy. To address this issue, a cyclicRGD peptide and an organic dye were covalently conjugated onto humanserum albumin (HSA), and these conjugates were subjected to fluorescenceimaging and histologic analysis, which confirmed the enhancedperformance in vivo (Chen et al., Mol. Imaging, 2009, 8:65-73). Thesuccess of this approach can be extended to other peptide-based probesand drugs that are physically conjugated with HSA for prolonged tumorpenetration and improved pharmacokinetics.

In some embodiments, the fusion polymers described herein comprise acollagen-binding domain and an HSA-binding domain. These fusion peptidescan directly bind to human serum albumin, which can in turn bind to avariety of therapeutic/biologic agent, and target thetherapeutic/biologic agent bound by HSA to neoplastic lesions. In someembodiments, the fusion polymers bind human serum albumin in anon-covalent manner (see Albumin-Aptamer 1 and 2 of FIG. 2E). Forexample, the HSA-binding domain can have the amino acid sequence of SEQID NO: 39, or SEQ ID NO: 40, or variants of these sequences. Skilledpractitioners will appreciate that the sequence of SEQ ID NO:39 or SEQID NO: 40 could be modified and tested to modify or retain at least someportion of its Fc binding activity and, accordingly, variants (i.e.,sequences with at least 70%, e.g., at least 80% or at least 90% identityto SEQ ID NO:15) can be useful in certain embodiments.

In some embodiments, the fusion polymers bind human serum albumincontain a thiol-reactive maleimide group, which is capable of bindingcovalently to the single free cysteine residue (Cys-34) of human serumalbumin (see Albumin-Aptamer 3 and 4 of FIG. 2E).

In some embodiments, the tumor-targeting and HSA-binding fusion polymersfurther include one or more linkers/spacers, to reduce the sterichindrances between the functional binding domains. The tumor-targetingand HSA-binding fusion polymers can have one or more linkers comprisingan amino acid sequence selected from the group consisting of SEQ ID NO:31, and SEQ ID NO: 9. In some embodiments, the fusion polymers areacetylated, amidated and/or PEGylated at N- or C-terminus. Inclusion ofD-amino acids, acetylation, amidation, and/or PEGylation increase thestability of the fusion polymers and make them more resistant toproteolysis.

Exemplary amino acid sequences of tumor-targeting and HSA-binding fusionpolymers include SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ IDNO: 44. Skilled practitioners will appreciate that variations in suchsequences may be possible, while retaining tumor- and HSA-bindingactivity. Accordingly, sequences having at least 70%, e.g., at least80%, 90%, 95%, or at least 99%, identity to SEQ ID NO: 41, SEQ ID NO:42, SEQ ID NO: 43, or SEQ ID NO: 44, can be utilized.

Tumor-Targeting and Paclitaxel-Binding Fusion Polymers with N-Terminalor C-Terminal Shielding

In addition to the collagen-binding and drug-binding domains describedabove, fusion polymers can be further modified to include a shieldinggroup on the terminus of the polymer. Non-limiting examples of these N-and C-terminal modifications are the addition of a PEG (poly(ethyleneglycol)) moiety or a maleimide group to link covalently with albumin viahuman serum albumin (HSA) Cys-34. Both of these additions may shield thepeptide from degradation, increase the stealth of the peptide andtherefore may improve the circulating half-life of the peptides. Thecovalent tethering of HSA may serve to resist proteolytic degradation byserum proteases and additionally may reduce filtration of the targetingpolymer by the kidneys. The addition of a maleimide group enablessite-specific covalent coupling of the thiol-reactive group with a freecysteine residue, wherein, for example, the free cysteine residue couldbe that of the albumin protein or attached to the end of an aptamer toallow for site-specific covalent linking Additional C-terminalmodifications include protease-resistant “caps”. These caps are D-aminoacid substituted “caps” on the terminus of the polymer that may inhibitproteolysis of the aptamers by amino-peptidases and carboxypeptidasespresent in serum. These D-amino acid substituted “caps” are denotedherein by three letter coded amino acids in the lower case or by “D-”followed by the three letter amino acid code.

In some embodiments, the fusion polymers bind human serum albumin in anon-covalent manner (see Albumin-Aptamer 1 and 2 of FIG. 2E). Forexample, the HSA-binding domain can have the amino acid sequence of SEQID NO: 39, SEQ ID NO: 40, or variants of these sequences. In someembodiments, the fusion polymers include a thiol-reactive maleimidegroup, which is capable of binding covalently to the single freecysteine residue (Cys-34) of human serum albumin (see Albumin-Aptamer 3and 4 of FIG. 2E).

In some embodiments, the tumor-targeting, paclitaxel-binding, andHSA-binding fusion polymers further include one or more linkers/spacers,to reduce the steric hindrances between the functional binding domains.The fusion polymers can have one or more linkers comprising an aminoacid sequence selected from the group consisting of SEQ ID NO: 31, andSEQ ID NO: 9. In some embodiments, the fusion polymers are acetylated,amidated and/or PEGylated at N- or C-terminus. Additionally, theinclusion of D-amino acids can increase the stability of the fusionpolymers and make them more resistant to proteolysis.

An exemplary amino acid sequence of a tumor-targeting,paclitaxel-binding, and PEG-shielded fusion polymer includes SEQ ID NO:27. Skilled practitioners will appreciate that variations in suchsequences may be possible, while retaining tumor- and paclitaxel-bindingand PEG-shielded activity. Accordingly, sequences having at least 70%,e.g., at least 80%, 90%, 95%, or at least 99%, identity to SEQ ID NO: 27can be utilized.

Tumor-Targeting Drug Delivery Nanoparticles (Nano-Tropins)

Provided herein are novel bifunctional fusion nanoparticles fortargeting pharmaceutical agents to pathological areas. The fusionnanoparticles described herein include two or more portions: (i) atleast one fusion polymer that includes a sequence that binds to theExposed Collagenous (XC-) proteins, and an amphiphilic polymer withdistinct hydrophobic and hydrophilic domains that can self-assemble withsimilar polymers to assemble a core-shell structure and (ii) ananoparticle (e.g., a micelle or liposome) that non-covalentlysequesters chemotherapeutic or biologic agents (e.g., FIG. 14A), forexample agents with hydrophobic or hydrophilic characteristics ornucleic acids (e.g., FIGS. 14B & 14C). These fusion polymers can alsoinclude (iii) linker segments and/or (iv) flanking sequences to improvethe functionality, pharmacokinetics, stability, and/or pharmacodynamicsof the targeted drug delivery. These fusion nanoparticles may enabletumor-targeted delivery of the chemotherapeutic and biologic agents,e.g., anticancer agents, and may be particularly suited for thetransport of drugs with low solubility.

In some embodiments, the drug-binding properties of these fusionnanoparticles are based on the non-covalent sequestering of the druginside a nanoparticle such as, but not limited to, a micelle orliposome. These nanoparticles can be made of amphiphilic polymers thatform a core-shell structure that is useful as a carrier for delivery ofdrugs as well as nucleic acids. These nanoparticles represent aversatile platform for cancer drug delivery due to their small size(10-100 nm), in vivo stability, prolonged blood circulation times,ability to transport insoluble-drugs, and ability to transport drugswithout substantially altering the chemical composition or commercialmanufacturing of the drug.

The fusion nanoparticles described herein can include at least onefusion polymer comprising (i) a collagen-binding domain and (ii) anamphiphilic polymer with distinct hydrophobic and hydrophilic blockdomains that self-assembles with similar polymers to assemble acore-shell structure. In some embodiments of the fusion nanoparticlesdescribed herein, the at least one fusion polymer is at least or about10% (e.g., at least or about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%,80%, or at least or about 90%) of the total number of polymers thatcomprise the micelle. In some embodiments, the at least one fusionpolymer is between about 10% and 50% of the total number of polymersthat comprise the micelle.

In some embodiments, the tumor-targeting drug-delivery fusionnanoparticles can further include one or more linkers/spacers, to reducethe steric hindrances between the functional domains. The fusionnanoparticles can have one or more linkers comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 22, SEQ ID NO:7, SEQ ID NO: 23, SEQ ID NO: 17, SEQ ID NO: 24, SEQ ID NO: 25, SEQ IDNO: 31, SEQ ID NO: 26, and SEQ ID NO: 9. In some embodiments, the fusionpolymers are acetylated, amidated and/or PEGylated at N- or C-terminus.Inclusion of D-amino acids, acetylation, amidation, and/or PEGylationmay increase the stability of the fusion polymers and make them moreresistant to proteolysis.

In some embodiments, the fusion nanoparticles function in the deliveryof drugs including but not limited to hydrophobic drugs, hydrophilicdrugs, and nucleic acids. In some embodiments, these drugs are Taxanes(e.g., Docetaxel, Paclitaxel), Doxorubicin, epirubicin, Platinum Drugs(Cisplatin, CDDP, DACHPt), R547 (a cyclin-dependent kinase inhibitor),TGX-221 (PI-3 kinase inhibitor), Captothecin, Gemcitabine, 5-fluouracil,Rifampicin, Tamoxifen, Ellipticin, ethotrexate, Daunomycin, estrogen,Curcumin, and various therapeutic siRNAs (see Jhaveri and Torchilin,Multifunctional polymeric micelles for delivery of drugs and siRNA April2014. Frontiers in Pharma. Vol 5. Art 77; Bennet and Kim, PolymerNanoparticles for Smart Drug Delivery 2014. Application of Nano. In DrugDelivery Ch 8 pages 257-310; and Kim et al., Engineered Polymers forAdvanced Drug Delivery March 2009. Eur J Pharm Biopharm. 71(3):420-430.which are incorporated herein in their entirety).

Generation of the Bifunctional Fusion Polymers

A fusion polymer described herein can be produced by expression of arecombinant nucleic acid encoding the polymer or by chemical synthesis(e.g., by solid-phase synthesis or other methods well known in the art,including synthesis with an ABI peptide synthesizer; Applied Biosystems,Foster City, Calif.). For example, a fusion polymer can be produced byexpression of a nucleic acid encoding the protein in prokaryotes. Theseinclude but are not limited to microorganisms such as bacteriatransformed with recombinant bacteriophage DNA, plasmid DNA or cosmidDNA expression vectors encoding a fusion protein of the invention. Theconstructs can be expressed in E. coli in large scale for in vitroassays. Purification from bacteria is simplified when the sequencesinclude tags for one-step purification by nickel-chelate chromatography.The construct can also contain a tag to simplify isolation of the fusionpolymer. For example, a polyhistidine tag of, e.g., six histidineresidues, can be incorporated at the amino terminal end of thefluorescent protein. The polyhistidine tag allows convenient isolationof the protein in a single step by nickel-chelate chromatography. Thefusion polymer described herein can also be engineered to contain acleavage site to aid in protein recovery. Alternatively, the fusionpolymers described herein can be expressed directly in a desired hostcell for assays in situ.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate co-precipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransfected with DNA sequences encoding the fusion polymer of theinvention, and a second foreign DNA molecule encoding a selectablephenotype, such as the herpes simplex thymidine kinase gene. Anothermethod is to use a eukaryotic viral vector, such as simian virus 40(SV40) or bovine papilloma virus, to transiently infect or transformeukaryotic cells and express the protein. (Eukaryotic Viral Vectors,Cold Spring Harbor Laboratory, Gluzman ed., 1982). Preferably, aeukaryotic host is utilized as the host cell as described herein.

Eukaryotic systems, and preferably mammalian expression systems, allowfor proper post-translational modifications of expressed mammalianproteins to occur. Eukaryotic cells which possess the cellular machineryfor proper processing of the primary transcript, glycosylation,phosphorylation, and, advantageously secretion of the gene productshould be used as host cells for the expression of fluorescentindicator. Such host cell lines may include but are not limited to CHO,VERO, BHK, HeLa, COS, MDCK, Jurkat, HEK-293, and WI38.

For long-term, high-yield production of recombinant proteins, stableexpression can be used. Rather than using expression vectors whichcontain viral origins of replication, host cells can be transformed withthe cDNA encoding a fusion protein of the invention controlled byappropriate expression control elements (e.g., promoter, enhancer,sequences, transcription terminators, polyadenylation sites, etc.), anda selectable marker. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. For example, following theintroduction of foreign DNA, engineered cells may be allowed to grow for1-2 days in an enriched media, and then are switched to a selectivemedia. A number of selection systems may be used, including but notlimited to the herpes simplex virus thymidine kinase (Wigler, et al.,Cell, 11:223, 1977), hypoxanthine-guanine phosphoribosyltransferase(Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA, 48:2026, 1962), andadenine phosphoribosyltransferase (Lowy, et al., Cell, 22:817, 1980)genes can be employed in tk⁻, hgprt⁻ or aprt⁻ cells respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler, et al., Proc.Natl. Acad. Sci USA, 77:3567, 1980; O'Hare, et al, Proc. Natl. Acad.Sci. USA, 8:1527, 1981); gpt, which confers resistance to mycophenolicacid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072, 1981; neo,which confers resistance to the aminoglycoside G-418 (Colberre-Garapin,et al., J. Mol. Biol., 150:1, 1981); and hygro, which confers resistanceto hygromycin (Santerre, et al., Gene, 30:147, 1984) genes. Recently,additional selectable genes have been described, namely trpB, whichallows cells to utilize indole in place of tryptophan; hisD, whichallows cells to utilize histinol in place of histidine (Hartman &Mulligan, Proc. Natl. Acad. Sci. USA, 85:8047, 1988); and ODC (omithinedecarboxylase) which confers resistance to the ornithine decarboxylaseinhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue L., In:Current Communications in Molecular Biology, Cold Spring HarborLaboratory, ed., 1987).

Techniques for the isolation and purification of either microbially oreukaryotically expressed polymers of the invention may be by anyconventional means such as, for example, preparative chromatographicseparations and immunological separations such as those involving theuse of monoclonal or polyclonal antibodies or antigen.

Generation of the Tumor-Targeted Drug-Delivery Nanoparticles

Fusion-polymers described herein for construction of a tumor-targeteddrug-delivery nanoparticle can be produced by the covalent linkage of atumor targeting polypeptide to a maleimide-activated polymer withdistinct hydrophobic and hydrophilic block domains. The tumor targetingpolypeptide, di- or multi-block polymer, and maleimide-activateddi-block copolymer can separately be produced by expression of arecombinant nucleic acid encoding the polymer or by chemical synthesisas described above. The tumor targeting aptamer sequence can furthercomprise one or more linkers. These linkers can be separately producedby expression of a recombinant nucleic acid encoding the polymer or bychemical synthesis or can be expressed with the recombinant polymer, asdescribed previously. One of the one or more linkers can be attached onthe N- or C-terminal end of the aptamer and further comprise a cysteineresidue such that the cysteine residue is the terminal residue. The di-or multi-block polymer can be produced to similarly comprise one or morelinkers. One of the one or more linkers of the polymer can be linked tothe end of the polymer that is adjacent to the hydrophilic block and canconnect a maleimide-group to the polymer to create themaleimide-activated polymer. This maleimide-group can react with thethiol group of the cysteine residue on the tumor-targeting peptide tosite-specifically form a covalent bond between the tumor targetingaptamer and the polymer. The covalent linkage of the tumor-targetingpeptide and the di- or multi-block creates a fusion polymer. This fusionpolymer can comprise the tumor-targeting domain, one or more linkers,the hydrophilic and hydrophobic blocks in such a structure as to havethe propensity to assemble into a hydrophilic shell surrounding ahydrophobic core or bilayer as described herein. The linking of thetumor-targeting peptide to the di- or multi-block polymer is such thatwhen the nanoparticle is assembled, the tumor-targeting domain of thefusion polymer extends outwardly from the hydrophilic surface of thenanoparticle such that the tumor-targeting domain can bind to XCproteins while associated with the nanoparticle.

Methods of Use of the Bifunctional Fusion Polymers and Nanoparticles

The fusion polymers and pharmaceutical compositions described herein canbe useful for the treatment of a disease, for example, cancer. Thecancer can be, e.g., a primary or metastatic cancer, including but notrestricted to, colorectal cancer, breast cancer, brain tumors, non-smallcell lung cancer, pancreatic cancer, prostate cancer, sarcoma,carcinoma, and/or melanoma. The cancer can be, e.g., cancer of thestomach, colon, rectum, mouth/pharynx, esophagus, larynx, liver,pancreas, lung, breast, cervix uteri, corpus uteri, ovary, prostate,testis, bladder, skin, bone, kidney, brain/central nervous system, head,neck and/or throat; sarcoma, or choriocarcinoma. In general, the methodsof treating cancer can include administering to a subject having canceran amount of the fusion polymer or pharmaceutical composition sufficientto treat cancer in the patient. An exemplary method of treating cancerin a subject using the fusion polymers can include: (a) providing ananti-cancer agent; (b) providing a bifunctional fusion polymer thatincludes: (i) sequences that bind to the anti-cancer agent, and (ii)sequences that bind to the exposed collagens present in cancerouslesions; (c) mixing the anti-cancer agent with the bifunctional fusionpolymer at a specific ratio under desired conditions for a sufficientlylong period to allow association of the fusion polymer with theanti-cancer agent, thereby forming an anticancer agent/fusion polymercomplex; and (d) administering to a subject in need of treatment aneffective amount of a pharmaceutical composition comprising theanticancer agent/fusion polymer complex. The conditions for the mixingstep can be optimized based on (i) the solubility and stability of theanticancer agent, (ii) the solubility and stability of the fusionpolymer, (iii) the N-terminal and C-terminal modifications of the fusionpolymers. For example, the mixing step can be performed in phosphatebuffered saline (PBS) at 30° C. for 30 min. In some embodiments, thesubject is a human. In some embodiments, the cancer is a solid tumor,for example, a sarcoma, a carcinoma, or a melanoma.

An exemplary method of treating cancer in a subject using the fusionnanoparticles can include: (a) providing an anti-cancer agent; (b)providing a bifunctional fusion nanoparticle that can sequester theanti-cancer agent and that comprises at least one bifunctional fusionpolymer that can bind to the exposed collagens present in cancerouslesions; (c) mixing the anti-cancer agent with the bifunctional fusionnanoparticle at a specific ratio under desired conditions for asufficiently long period to allow association of the fusion nanoparticlewith the anti-cancer agent, thereby forming an anticancer agent/fusionnanoparticle complex; and (d) administering to a subject in need oftreatment an effective amount of a pharmaceutical composition comprisingthe anticancer agent/fusion nanoparticle complex. The conditions for themixing step can be optimized based on (i) the solubility and stabilityof the anticancer agent, (ii) the solubility and stability of the fusionnanoparticle, and (iii) the N-terminal and C-terminal modifications ofthe fusion polymers (see Kim et al., 2009, Eur J. Pharm Biopharm,71:420-430; Sutton et al., 2007; Lee et al., 2006; Du and O'Reilly,2009, Royal Soc Chem, Soft Matter 5:3544-3561; Huertas et al., 2010, IntJ. Pharmaceutics, 385:113-142; Hu et al., 2014, Royal Soc Chem. DOI:10.1039/c3nr05444f; Bennet and Kim, 201, Intech.http://dx.doi.org/10.5772/58422 which are incorporated herein in theirentirety). In some embodiments, the subject is a human. In someembodiments, the cancer is a solid tumor, for example, a sarcoma, acarcinoma, or a melanoma.

The fusion polymers and nanoparticles described herein can also be used,e.g., for imaging and/or tracking primary and/or metastatic tumors in asubject. For example, the fusion polymers and nanoparticles can belabeled with a detectable moiety, such as a radioactive isotope, amagnetic compound, an x-ray absorber, a fluorescent molecule, a chemicalcompound, and/or a biological tag. After administering the labeledfusion polymer or nanoparticle to a subject, the fusion polymers andnanoparticles are targeted to the primary and metastatic tumors in thesubject through its collagen binding domain. Tumors can then be detectedusing computed tomography, radiography, magnetic resonance imaging,laser scanning microscopy, immunohistochemistry, fluorescent microscopy,Raman spectroscopy, optical coherence tomography (OCT), detection ofradiation (e.g., x-ray) scattering or absorption, ultrasound, and/orisotope detection. Practitioners will appreciate that determining thedose to be administered to the subject for imaging or tracking is withinthe skill of the practitioner and will depend upon the type and locationof the tumor(s) in the patient, the type of detectable moiety to beused, and the type of imaging to be performed.

Pharmaceutical Compositions, Dosage Regimen, and Methods ofAdministration

Provided herein are also pharmaceutical compositions comprising one ormore of the fusion polymers or nanoparticles described herein. Thecompositions can further include one or more therapeutic and/or biologicagents known in the art to be effective in treating cancer, i.e., ananti-cancer agent. Such pharmaceutical compositions can be used to treatcancer as described above. In some embodiments, the pharmaceuticalcomposition is administered to a subject in need of treatmentintravenously or subcutaneously.

The active ingredient of a pharmaceutical composition can be formulatedfor delivery by any available route including, but not limited toparenteral (e.g., intravenous), intradermal, subcutaneous, oral, nasal,bronchial, ophthalmic, transdermal (topical), transmucosal, rectal, andvaginal routes. A pharmaceutical composition provided herein can includeanother delivery agent and a pharmaceutically acceptable carrier. Asused herein the term “pharmaceutically acceptable carrier” includessolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. Supplementary activecompounds can also be incorporated into pharmaceutical formulations thatcontain an antibody or antigen-binding fragment thereof as describedherein.

Methods of formulating suitable pharmaceutical compositions are known inthe art, see, e.g., Remington: The Science and Practice of Pharmacy,21st ed., 2005; and the books in the series Drugs and the PharmaceuticalSciences: a Series of Textbooks and Monographs (Dekker, N.Y.). Forexample, solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerin, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injection can include sterileaqueous solutions (where water soluble), dispersions, and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.

Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent that delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying, which yield a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orsterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds can be delivered in theform of an aerosol spray from a pressured container or dispenser thatcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer. Such methods include those described in U.S. Pat. No.6,468,798.

Certain tumors may be accessible by administration by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

In some embodiments, the therapeutic compounds can be prepared withcarriers that will protect the therapeutic compounds against rapidelimination from the body, such as a controlled release formulation,including implants and microencapsulated delivery systems.

In some embodiments, the pharmaceutical composition can be directlyadministered to the areas of active angiogenesis. In some embodiments,the pharmaceutical composition can be administered through conventionalroutes, e.g., intravenously. Microencapsulation technology or liposomescan be used to protect the pharmaceutical compositions duringcirculation and release them at the site of active angiogenesis.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The therapeutic and/or biologic agents can be administered in aneffective amount, at dosages and for periods of time necessary toachieve the desired result. An effective amount can be administered inone or more administrations, applications or dosages. A therapeuticallyeffective amount of a pharmaceutical composition (i.e., an effectivedosage) depends on the pharmaceutical composition selected. Thecompositions can be administered from one or more times per day to oneor more times per week; including once every other day. The skilledartisan will appreciate that certain factors may influence the dosageand timing required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of the pharmaceutical compositions described herein caninclude a single treatment or a series of treatments.

Dosage regimens can be adjusted to provide the optimum therapeuticresponse. For example, several divided doses can be administered dailyor the dose can be proportionally reduced as indicated by the exigenciesof the therapeutic situation. Those skilled in the art will be aware ofdosages and dosing regimens suitable for administration of the newmonoclonal antibodies disclosed herein or antigen-binding fragmentsthereof to a subject. See e.g., Physicians' Desk Reference, 63rdedition, Thomson Reuters, Nov. 30, 2008. For example, Dosage, toxicityand therapeutic efficacy of the therapeutic compounds can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit high therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

Kits

Also provided are kits that include one or more of the fusion polymersdescribed herein. Kits generally include the following major elements:packaging, reagents comprising binding compositions as described above,optionally a control, and instructions. Packaging can be a box-likestructure for holding a vial (or number of vials) containing saidbinding compositions, a vial (or number of vials) containing a control,and instructions for use in a method described herein. Individualsskilled in the art can readily modify the packaging to suit individualneeds.

In some embodiments, a kit provided herein can include at least one(e.g., one, two, three, four, five, or more) composition containing atleast one (e.g., one, two, three, four, five, or more) of the fusionpolymers described herein, and at least one (e.g., one, two, three,four, five, or more) other composition in a separate vial containing atherapeutic or biologic agent known in the art to be effective intreating cancer.

Compositions and kits as provided herein can be used in accordance withany of the methods (e.g., treatment methods) described above. Forexample, compositions and kits can be used to treat cancer. Thoseskilled in the art will be aware of other suitable uses for compositionsand kits provided herein, and will be able to employ the compositionsand kits for such uses.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1 Optimization and Analysis of Collagen-Binding Sequences

Three fluorescein isothiocyanate (FITC)-labeled collagen-bindingconstructs were generated and tested. Construct 1 (ac-L1/XC-BD1/L3/FITC)had the sequence ofAc-R-R-G-V-H-V-G-W-R-E-P-S-F-M-A-L-S-M-P-H-G-G-S-G-K-(FITC) (SEQ ID NO:45); Construct 2(ac-L1/XC-BD2/L3/FITC) had the sequence ofAc-R-R-G-V-H-V-G-W-R-E-P-G-R-M-E-L-N-M-P-H-G-G-S-G-K-(FITC) (SEQ ID NO:46); Construct 3 (ac-L2/XC-BD2/L3/FITC) had the sequence ofAc-R-R-G-V-R-V-A-W-R-E-P-G-R-M-E-L-N-M-P-H-G-G-S-G-K-(FITC) (SEQ ID NO:47). As shown in FIG. 3A, strategic modifications were made in thecollagen-binding domains (XC-BD), the flanking domains, and therespective linkers. Increasing amounts of these constructs were appliedto the chromatography columns of Collagen-Agarose beads, followed bysuccessive washes with (1) PBS, (2) PBS/PBS, Tween-20, BSA. Theretention of the constructs to the column was documented by use of ablue-light transilluminator with an amber filter; quantitative data wasobtained with a Quantus fluorometer. FIG. 3B shows that both Construct 1and Construct 2 bound to collagen in a dose dependent manner in vitroand were selected for further characterization in vivo. FIGS. 3C-3D showthat Construct 2 was efficiently targeted to subcutaneous tumorsfollowing intravenous infusion into the tail vein of nude mice.

Example 2 Generation and Test of Tumor-Targeting and Paclitaxel-BindingFusion Polymers (Taxol-Tropins)

Three tumor-targeting and paclitaxel-binding fusion polymers(Taxol-Apatmers or Tx-Apt) were generated and tested. Tx-Apt1(XC-BD1/TxBD1/PEG) had the sequence ofAc-R-R-G-V-H-V-G-W-R-E-P-S-F-M-A-L-S-M-P-H-G-G-S-G-R-G-V-G-I-M-K-A-C-G-R-T-R-V-T-S-A-G-S-G-(mPEG)(SEQ ID NO: 48). Tx-Apt2 (Daa-XC-BD2/TxBD2-Daa, lower case letters areD-isomers) had the sequence ofAc-[r-r-G-V-H-V-G]-W-R-E-P-G-R-M-E-L-N-M-P-H-[G-G-S-G]-R-G-V-G-I-M-K-A-C-G-R-T-R-H-T-V-R-m-G(SEQ ID NO: 49). Tx-Apt3 (PEG/XC-BD2/TxBD3-Daa, lower case letters areD-isomers) had the sequence ofmPEG-K-G-R-R-G-V-H-V-G-W-R-E-P-G-R-M-E-L-N-M-P-H-G-G-S-G-R-G-V-G-I-M-R-A-C-G-R-T-R-H-T-V-R-m-G(SEQ ID NO: 50). The structures of three Tx-Apts were showndiagrammatically in FIG. 4A.

The Tx-Apts was tested by collagen-agarose column chromatography. Either100 ug or 300 ug of these Tx-Apts and Oregon Green-488-labeledpaclitaxel were applied to the chromatography columns ofcollagen-agarose beads, followed by successive washes with (1) PBS, (2)PBS/PBS, Tween-20, BSA. The retention of the constructs to the columnwas documented by use of a blue-light transilluminator with an amberfilter; quantitative data was obtained with a Quantus fluorometer. FIG.4B shows all three Tx-Apts successfully delivered OregonGreen-488-labeled paclitaxel to collagen, and the Tx-Apt2 achieved thebest delivery of paclitaxel. Without Tx-Apt, the OregonGreen-488-labeled paclitaxel alone did not bind to collagen (Control),illustrating Tx-Apt-dependent paclitaxel binding to collagen (FIG. 4B).The serum stability (resistance to proteolysis) of these Tx-Apts overtime was shown in FIG. 4C, Tx-Apt2 was the most stable of the threeTx-Apts.

Taxol-aptamers were tested for biological activity in cancer cells invitro. Collagen-agarose column chromatography was first used to verifythat the binding of Taxol to collagen beads was indeed peptide-dependent(FIG. 5C). Human MDA-MB-231 breast cancer cells were plated onto thewashed Control and Taxol-Aptamer beads and incubated for one day. TheMDA-MB-231 breast cancer cells on Taxol-Aptamer beads showed theclassical signs of cell death and destruction, that is, a mitotic blockfollowed by apoptosis, necrosis, and cytolysis (FIG. 5B, arrows). TheMDA-MB-231 breast cancer cells on Control collagen beads readily adheredand proliferated (FIG. 5A, dashed circles), spreading out along the XCsurfaces of the bead matrix at 48 and 72 hours. Thus, thepaclitaxel/Taxol-Aptamer complex was cytotoxic to MDA-MB-231 breastcancer cells when bound to XC protein matrix, indicating retention ofthe biologic activity of paclitaxel.

Targeting of paclitaxel by Taxol-aptamers to tumor was examined in vivo.Nude mice bearing subcutaneous human pancreatic cancer xenografts wereintravenously injected with fluorescent Oregon Green-488 paclitaxel inthe presence or absence of the Taxol-aptamers (FIG. 6A). The xenografttumors were excised and analyzed by fluorescence imaging, which showedthe Taxol-aptamer-dependent tumor targeting of paclitaxel (FIG. 6B).Moreover, when used with Taxol-aptamer, paclitaxel exhibitedanti-angiogenic activity (i.e., potential efficacy) at 0.2 mg/kg, only1/50^(th) of the normal effective dose of paclitaxel in mice (10 mg/kg)(FIG. 6C). The anti-angiogenic activity seen in targeted paclitaxeltreated mice is 1/10th of the low “metronomic” doses commonly used.These data show Oregon Green-labeled paclitaxel was targeted/deliveredto subcutaneous tumors in nude mice in a Taxol-aptamer dependent manner.

The specificity of the Taxol-Tropins for XC-proteins was furtherdemonstrated in vitro utilizing tumor-targeting taxol-binding Tx-Aptamer2 (see FIG. 4A) and Taxol-Green (see FIG. 16A and FIG. 16B). Atri-layered matrix of Sepharose/XC-Agarose/Sepharose (0.5 ml each) and acontrol column of 1.5 ml Sepharose 4B (agarose-blank) were prepared. Amixture of fluorescent Taxol-Green (5 ug) and Tx-Aptamer 2 (250 ug) in 1ml PBS was incubated for 30 minutes at room temperature before beingapplied to the PBS-equilibrated column. A control mixture containedTaxol-Green minus Tx-Aptamer 2. After application of the Taxol-greenmixtures, the columns were washed consecutively with PBS, followed byPBS-T (0.05% Tween-20), followed by 0.5 M NaCl in PBST, followed by 1.0M PBST. Photos of the resulting columns were obtained under bright fieldlighting (see FIG. 16A) and blue-light/amber filter (see FIG. 16B).

In this example the column washes represent the systemic circulation,the Sepharose-blanks represent normal (non-diseased) tissues, and theXC-Agarose represents tumor tissues. The bright band seen under the bluelight in the XC-agarose and not in the control column is a graphicdemonstration of XC-protein selectivity (see FIG. 16B), i.e., asimulation of tumor targeting in vitro. Additionally the Tx-Aptamer2/Taxol Green fusion complex remained tightly bound to the XC-proteinmatrix after stringent washing, demonstrating the affinity of theTaxol-Tropin complexes for XC-proteins.

Example 3 Generation and Test of Tumor-Targeting and mAb-Binding FusionPolymers (mAb-Tropins)

FIG. 7A shows the structures of four immunoglobulin-binding,collagen-binding fusion peptides that were generated and tested. ThemAb-Aptamer 1 (XC-BD1/mAb-BDx2) had the sequence ofR-R-G-V-H-V-G-W-R-E-P-S-F-M-A-L-S-M-P-H-G-G-G-G-G-H-W-R-G-W-V-G-G-G-G-G-H-W-R-G-W-V(SEQ ID NO: 51). The mAb-Aptamer 2 (Daa-XC-BD2/mAb-BDx2-Daa, lower caseletters are D-isomers) had the sequence ofr-r-G-V-H-V-G-W-R-E-P-G-R-M-E-L-N-M-P-H-G-G-S-G-G-H-W-R-G-W-V-A-G-G-S-G-G-H-W-R-G-W-V-a-a(SEQ ID NO: 52). mAb-Aptamer 3 (PEG/XC-BD2/mAb-BDx2-Daa) had thesequence of(PEG)-C-G-R-R-G-V-H-V-G-W-R-E-P-G-R-M-E-L-N-M-P-H-G-G-S-G-G-H-W-R-G-W-V-A-G-G-S-G-G-H-W-R-G-W-V-a-a(SEQ ID NO: 53). mAb-Aptamer 4 (PEG/XC-BD2/mAb-BDx2-Daa) had thesequence of(PEG)-[C-A-R-R-G-V-H-V-G]-W-R-E-P-G-R-M-E-L-N-M-P-H-[G-G-S-G-G]-H-W-R-G-W-V-A-[G-G-S-G-G]-H-W-R-G-W-V-A-p-t(SEQ ID NO: 54).

The four mAb-aptamers were tested by collagen-agarose columnchromatography, which showed mAb-aptamer-dependent antibody (fluorescentRabbit-Anti-Human Abs) binding to collagen agarose beads (FIG. 7B).Among the four aptamers tested, mAb-Aptamer 2 and mAb-Aptamer 4 resultedhigher binding of antibodies to the collagen beads (FIG. 7B). The serumstability (resistance to proteolysis) of mAb-Aptamer 1, mAb-Aptamer 2and mAb-Aptamer 4 were further analyzed. The PEG-bearing mAb-Aptamer 4showed the greatest serum stability among the tested mAb-aptamers (FIG.7C).

Monoclonal antibody bevacizumab (Avastin) was conjugated to a greenfluorescent dye 488-Oregon Green, followed by size exclusionchromatography to isolate the labeled Avastin. Structures of tworepresentative mAb-Tropins (M03 and P38) were shown in FIG. 8A. Avastinwas incubated with M03 or P38 in PBS for 30 min at 30° C. and thenapplied to the chromatography column of collagen-agarose beads, followedby successive washes with PBS, and PBS/Tween-20, PBS/Tween-20/BSA. Theretention of Avastin to the collagen column was documented by use of ablue-light transilluminator with an amber filter; quantitative data wasobtained with a Quantus fluorometer. As shown in FIG. 8B, the combinedantibody-binding and collagen-binding activity of M03 was positive andM03-dependent, while that of P38 was nearly negative. Increasing theconcentrations of M03 also increased the collagen-bound retentates ofAvastin in the column (FIGS. 8BC-8D).

The effect of Avastin/mAb-Tropin on the VEGF-stimulated human veinendothelial cell (HUVEC) proliferation was investigated by passing theculture media through a collagen-agarose chromatography column. HumanVascular Endothelial Cells (HUVEC) were evenly plated in complete growthmedium (M200 with 2% serum plus EGF and FGF supplements) and allowed forattachment, growth and division. The cells were then starved in 0.2%serum without supplements for a four-hour period while thechromatography columns were prepared. Avastin/mAb-Tropin complex wasloaded onto collagen chromatography column to form VEGF-Trap(XC/mAb-Tropin/Avastin complex) columns. To test whether thecollagen-bound Avastin is capable of blocking the VEGF-stimulated HUVECproliferation, low-serum media was spiked with VEGF (10 ng/ml) and onehalf of this VEGF-containing medium was poured over either Controlcolumns or our VEGF-Trap (collagen/mAb-Tropin/Avastin complex) columns.The serum-starved cells were then fed with the respective column passthrough (eluates). The VEGF-stimulated growth and proliferation of theHUVEC cells were unaffected in the Control columns (FIGS. 9A-C) but wereblocked in the VEGF-Trap (collagen/mAb-Tropin/Avastin complex) columns(FIGS. 9D-F). The inhibition of VEGF-stimulated growth by VEGF-Trapcolumns indicated that Avastin in the collagen-bound/mAB-Tropin/Avastincomplexes was biologically active and had anti-angiogenic effects.

Example 4 Generation and Test of Tumor-Targeting and RNA-Binding FusionPolymers (RNA-Tropins)

FIG. 10A shows the structures of two RNA-binding and collagen-bindingfusion peptides that were generated and tested. The RNA-Apt 07(CPP-R9/XC-BD1) had the sequence of SEQ ID NO: 55. The RNA-Apt 08(XC-BD1/CPP-R9) had the sequence of SEQ ID NO: 35.

Short oligonucleotides labeled with Oregon Green 488 were incubated withincreasing amounts of RNA-Apt 07 or RNA-Apt 08 in PBS for 30 min at 30°C. and then applied to the chromatography column of collagen-agarosebeads, followed by successive washes with (1) PBS, (2) PBS/PBS,Tween-20, BSA. The retention to the column was documented by use of ablue-light transilluminator with an amber filter; quantitative data wasobtained with a Quantus fluorometer. Both RNA-Apt 07 and RNA-Apt 08(with polyarginine R9) depleted fluorescently labeled-oligonucleotidesfrom the eluates (FIG. 10B) while retaining the labeled oligonucleotideson the collagen-agarose mini-columns (FIG. 10C).

Example 5 Test of Tumor-Targeting Polypeptide Delivery of IgGs to Tumorsin Mice

A murine xenograft model of metastatic cancer was established usingsubcutaneous tumors composed of pancreatic cancer cells (MIA PaCa).Following the establishment of tumors, the mice were injected i.v. viathe tail vein and after 60 minutes they were imaged with bright fieldand fluorescence settings (see FIG. 11A-11C).

Mice injected with IgG^(FITC) without the collagen-bindingtumor-targeting aptamer demonstrated very little accumulation ofIgG^(FITC) in the tumor (See FIG. 11A). Conversely, when the mice wereinjected with IgG^(FITC) linked to a tumor-targeting aptamer theaccumulation of IgG was visible within the tumor (see FIG. 11B).Untreated mice were used to determine the level of backgroundfluorescence (see FIG. 11C for control). The accumulation of IgG^(FITC)within the tumor after i.v. injection is evidence of the ability of thetumor-targeting peptides to deliver the IgG directly to the site of thetumor.

Example 6 Generation and Test of Tumor-Targeting and Human Serum Albumin(HSA)-Binding Fusion Polymers

Two maleimide-activated tumor-targeting peptides, Albumin-Aptamer 3(Mal-PEG2-L1-XCBD2a) and Albumin-Aptamer 4 (aXCBD2-L2-PEG2-Mal)(structures shown in FIG. 2E), were generated and tested for theirability to bind both HSA and an XC-agarose column.

HSA was reduced with 3 molar excess DTT, followed by dialysis ordesalting into PBS to remove the remaining DTT. This concentration ofDTT selectively reduces Cys-34 of HSA. The two Albumin-Aptamerconstructs, Apt-3 or Apt-4, were incubated with the reduced HSAovernight at 4° C. The Albumin-Aptamer constructs were then tested bycollagen-agarose (XC) column chromatography (FIG. 12).

The XC-bound Albumin-Aptamer constructs were detected using FITC-labeledgoat anti-HSA antibodies. Incubation with labeled goat antibodies wasfollowed by stringent washing (PBST plus 2M NaCl). The XC-boundAlbumin-Aptamer constructs were documented by fluorescence photography,as seen in FIG. 12, using a blue-light transilluminator equipped with anamber filter and a Leica V-LUX1 digital camera.

By selectively reducing the Cys-34 of HSA, the maleimide activatedlinker successfully bound HSA to the collagen-binding aptamer. In FIG.12 the binding of Apt-3 (structure shown in FIG. 2E) appears to bestronger than that of Apt-4.

Example 7 Tumor-Targeting, Taxol-Binding, Albumin-Linked Fusion Polymers(TargaTaxel)

A structural schematic of the novel tripartite onco-aptamer,CpBio-MA3TX, named TargaTaxel, is shown in FIG. 13 and is an exampleusing the covalent binding of Albumin for protection and shielding ofthe peptide. This schematic diagrams the (i) maleimide-activated,HSA^(Cys-34) binding domain, (ii) the tumor-targeting domain consistingof a high-performance XC-binding domain, and (iii) an optimizedpaclitaxel-binding domain for the carrying of taxol. The tethering ofHSA to this taxol-binding and tumor-targeting fusion aptamer serves as a“shield” that resists proteolytic degradation by serum proteases. Thestable linkage to albumin additionally serves to reduce filtration ofthe targeting aptamer by the kidneys, thus extending the biologicalhalf-life and circulating time of the construct.

This increase in stability and circulating half-life of the constructscan be addressed by the addition of a maleimide group to the N-terminusor C-terminus to link covalently with albumin via HSA Cys-34 or by theaddition of a PEG moiety to the N-terminus or C-terminus, among others.These modifications serve to “shield” the constructs and permit a longerbiological half-life. Skilled practitioners will appreciate thatvariations in these constructs are possible and such variants may beuseful in the present invention.

Example 8 Generation and Test of Tumor-Targeting Nanoparticles

Nanoparticles are useful for in vivo drug delivery due to a longcirculating half-life and their ability to improve the solubility,pharmacokinetics, and resulting efficacy of their cargos, particularlyhydrophobic agents. By using the tumor-targeting peptide of the presentinvention linked to a drug-delivering nanoparticle, drugs can bedelivered selectively and efficiently to tumors, without substantiallyaltering the chemical composition or manufacturing of the drug.

Schematics of an exemplary nanoparticle tumor-targeting system are shownin FIG. 14A-14C. FIG. 14A is the structure of the maleimide-activateddi-block copolymer and a schematic of a bifunctional polymer formed bythe linking of a tumor-targeted collagen-binding aptamer and amaleimide-activated di-block copolymer. The maleimide-activated di-blockcopolymer includes a hydrophobic PDLLA block, a hydrophilic PEG blockand a maleimide group connected to the di-block by a linker. The tumortargeting aptamer schematic diagrams a protective D-amino acid cap onthe C-terminal for protease resistance, a XC-binding tumor targetingpeptide sequence, and a linker at the N-terminus connecting a terminalcysteine residue. The free-cysteine residue enables site-specificcovalent coupling to the activated maleimide link on the di-blockcopolymer creating a fusion polymer. This polymer is incorporated at aratio of at least or about ˜10% (at least or about 10%, 20%, 30%, 40%,50%, 70%, 90%, or 99%) of the total copolymers that assemble into themicelle nanoparticle.

A schematic for a drug loaded micelle comprising the di-block polymer isshown in FIG. 14B. FIG. 14C is a schematic of a tumor-targeted drugloaded micelle made with fusion polymers displaying a tumor-targetingaptamer on the surface of a micelle. This tumor-targeting sequencedirects the nanoparticle to seek out and accumulate in thediseased/cancerous tissues following intravenous infusion. Thus, thefusion nanoparticles described herein may make conventionalchemotherapeutic and biologic agents more efficient with great efficacywhile lessening unwanted side effects, improving the overall TherapeuticIndex and patient survival.

In vitro testing of the tumor-targeting nanoparticles was performedusing XC-agarose chromatography. FIG. 15A-15C are schematics, images andgraphs of a nanoparticle tumor-targeting system using coumarin-6 as amodel hydrophobic drug. A schematic of the two tumor-targetedcollagen-binding aptamers that were tested is shown in FIG. 15A.

Polymer micelles were prepared in H₂O by a solvent evaporation methodusing 5% PEG-PDLLA di-block polymers and 0.5% maleimide-activateddi-block polymers PEG-PLGA in DCM. Micelles were then filtrated (0.45u),diluted with PBS (1:1) and coupled overnight to the free cysteineresidue on the terminus of the tumor-targeting aptamer (see Kim et al.,2009, Eur J. Pharm Biopharm, 71:420-430; Sutton et al., 2007; Lee etal., 2006; Du and O'Reilly, 2009, Royal Soc Chem, Soft Matter5:3544-3561; Huertas et al., 2010, Int J. Pharmaceutics, 385:113-142; Huet al., 2014, Royal Soc Chem. DOI: 10.1039/c3nr05444f; Bennet and Kim,201, Intech. http://dx.doi.org/10.5772/58422 which are incorporatedherein in their entirety). This coupling reaction was performed in PBSfor 2-4 hours at room temperature or overnight at 4° C. or roomtemperature and resulted in the covalent linkage of the copolymerPEG-maleimide group to the targeting aptamer.

The resulting fusion nanoparticles were analyzed by XC-agarosechromatography. FIG. 15B are images of a XC-agarose chromatographyanalysis of polymeric micelles loaded with Courmarin-6 and coupled tothe tumor-targeting aptamers. The fusion nanoparticles exhibitedtumor-targeting peptide-dependent binding to the XC-agarose beads understringent washing conditions (FIG. 15B). Quantification of the columneluates confirmed the tumor-targeting peptide-dependent reduction ofCoumarin-6/nanoparticles in the eluates (FIG. 15C). Fusion nanoparticleswith tumor-targeting Aptamer 2 exhibited less efficient retention ofCoumarin-6 and binding to the XC-agarose beads than Aptamer 1. Thesedata successfully demonstrated the bifunctional tumor-targeting and drugdelivery nanoparticle system.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A fusion polymer comprising (i) a tumor targeting aptamer sequencethat binds to exposed collagenous (XC) proteins, and (ii) a drug bindingdomain that binds to a drug or biologic agent. 2.-3. (canceled)
 4. Thefusion polymer of claim 1, wherein the drug binding domain is an RNA,growth factor, immunoglobulin, chemotherapeutic agent or human serumalbumin binding domain, and the drug is an RNA, a growth factor, animmunoglobulin, a chemotherapeutic agent or human serum albumin,respectively. 5.-21. (canceled)
 22. A fusion polymer comprising (i) atumor targeting aptamer sequence that binds to exposed collagenous (XC)proteins, and (ii) a di-block copolymer comprising a hydrophilic blockand a hydrophobic block. 23.-32. (canceled)
 33. A fusion polymercomprising (i) a tumor targeting aptamer sequence that binds to exposedcollagenous (XC) proteins, and (ii) a multi-block polymer comprisingsequentially a first hydrophilic block, a first hydrophobic block, asecond hydrophobic block, and a second hydrophilic block. 34.-90.(canceled)
 91. A method of making the fusion polymer of claim 22,comprising: (a) providing the tumor-targeting domain that binds toexposed collagenous proteins and a first linker bound to the N-terminusof the tumor-targeting domain, and wherein a cysteine residue is boundto the N-terminus of the first linker; (b) providing the di-blockcopolymer comprising the hydrophilic block and the hydrophobic block,wherein a second linker is bound to the terminus of the hydrophilicblock and wherein a maleimide-group is bound to the second linkerterminus that is unbound to the hydrophilic block; and (c) combining (a)and (b) under conditions such that the maleimide-group of (b) reactswith the thiol group of the cysteine residue of (a) to form a covalentbond between (a) and (b), thereby creating the fusion polymer.
 92. Amethod of making a micelle, comprising: (a) providing a first and asecond plurality of di-block copolymers, each comprising a hydrophobicblock and a hydrophilic block, wherein the di-block copolymers of thesecond plurality each comprise a linker bound to the terminus of thehydrophilic block, and wherein a maleimide group is bound to the linkerterminus that is unbound to the hydrophilic block; (b) mixing the firstand second plurality of di-block copolymers to thereby form a micelle;(c) providing a tumor-targeting domain that binds to exposed collagenousproteins and that comprises a cysteine residue on a terminus of thedomain; and (d) combining the micelle of (b) with the tumor-targetingdomain of (c) under conditions such that a maleimide of the di-blockcopolymers of the second plurality in the micelle reacts with thecysteine residue of the tumor-targeting domain, thereby connecting thetumor-targeting domain and the micelle.
 93. A method of making amicelle, comprising: (a) providing the fusion polymer of claim 22 and aplurality of di-block copolymers, each comprising a hydrophobic blockand a hydrophilic block; (b) mixing the fusion polymer with theplurality of di-block copolymers to thereby form a micelle. 94.(canceled)
 95. A method of making a liposome, comprising: (a) providinga first and a second plurality of multi-block polymers, each comprisingsequentially a first hydrophilic block, a first hydrophobic block, asecond hydrophobic block, and a second hydrophilic block, wherein themulti-block polymers of the second plurality each comprise a linkerbound to the terminus of the first hydrophilic block, and wherein amaleimide group is bound to the linker terminus that is unbound to thehydrophilic block; (b) mixing the first and second plurality ofmulti-block polymers to thereby form a liposome; (c) providing atumor-targeting domain that binds to exposed collagenous proteins andthat comprises a cysteine residue on a terminus of the domain; and (d)combining the liposome of (b) with the tumor-targeting domain of (c)under conditions such that a maleimide of the multi-block polymers ofthe second plurality in the liposome reacts with the cysteine residue ofthe tumor-targeting domain, thereby connecting the tumor-targetingdomain and the liposome.
 96. A method of making a liposome, comprising:(a) providing the fusion polymer of claim 33 and a plurality ofmulti-block polymers, each comprising sequentially a first hydrophilicblock, a first hydrophobic block, a second hydrophobic block, and asecond hydrophilic block; (b) mixing the fusion polymer with theplurality of multi-block polymers to thereby form a liposome. 97.(canceled)
 98. A micelle comprising a hydrophobic interior and ahydrophilic surface and at least one fusion polymer according to claim22, wherein the tumor targeting domain of the at least one fusionpolymer extends outwardly from the hydrophilic surface of the micelle,such that the tumor targeting domain can bind to XC proteins whileassociated with the micelle.
 99. A drug-delivery nanoparticle comprising(i) the micelle of claim 98; and (ii) a drug sequestered in thehydrophobic interior of the micelle.
 100. A liposome comprising a lipidbilayer having a hydrophilic exterior surface and an aqueous interiorand comprising at least one fusion polymer according to claim 33,wherein the tumor targeting domain of the at least one fusion polymerextends outwardly from the hydrophilic exterior surface of the liposome,such that the tumor targeting domain can bind to XC proteins whileassociated with the liposome.
 101. A drug-delivery nanoparticlecomprising (i) the liposome of claim 100 and (ii) a drug sequesteredinside the aqueous interior of the liposome.
 102. A drug-deliverynanoparticle comprising (i) the liposome of claim 100 and (ii) a drugsequestered inside the lipid bilayer of the liposome.
 103. A method oftreating a tumor in a subject, the method comprising: administering to asubject in need of such treatment a pharmaceutical compositioncomprising the fusion polymer of claim 1 and a pharmaceuticallyacceptable carrier in an amount sufficient to treat the tumor.
 104. Amethod of treating a tumor in a subject, the method comprising:administering to a subject in need of such treatment a pharmaceuticalcomposition comprising the drug-delivery nanoparticle of claim 99 and apharmaceutically acceptable carrier in an amount sufficient to treat thetumor.
 105. A method of treating a tumor in a subject, the methodcomprising: administering to a subject in need of such treatment apharmaceutical composition comprising the drug-delivery nanoparticle ofclaim 101 and a pharmaceutically acceptable carrier in an amountsufficient to treat the tumor.
 106. A method of treating a tumor in asubject, the method comprising: administering to a subject in need ofsuch treatment a pharmaceutical composition comprising the drug-deliverynanoparticle of claim 102 and a pharmaceutically acceptable carrier inan amount sufficient to treat the tumor.
 107. A method of treatingcancer in a subject, the method comprising: administering to a subjectin need of such treatment a pharmaceutical composition comprising thefusion polymer of claim 1 and a pharmaceutically acceptable carrier inan amount sufficient to treat the tumor.
 108. A method of treatingcancer in a subject, the method comprising: administering to a subjectin need of such treatment a pharmaceutical composition comprising thedrug-delivery nanoparticle of claim 99 and a pharmaceutically acceptablecarrier in an amount sufficient to treat the tumor.